Jack bean urease (EC 3.5.1.5). V. On the mechanism of
action of urease on urea, forrnamide, acetamide,
N-methylurea, and related compounds1
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
Depurtrnetrt of Biochemistry, Utliversity of' Q~reet~slund,
St. Lucicr, A~lstraliu4067
Received April 26, 1 9794
Dixon, N. E., Riddles, P. W., Gazzola, C., Blakeley, R. L. & Zerner, B. (1980) Jack bean urease
(EC 3.5.1.5). V. On the mechanism of action of urease on urea, forrnarnide, acetarnide,
N-rnethylurea, and related compounds. Cutr. J. Biochern. 58, 1335-1344
Acetarnide and N-methylurea have been shown for the first time to be substrates for jack bean
urease. In the enzymatic hydrolysis of urea, forrnarnide, acetarnide, and N-rnethylurea at pH
7.0 and 38"C, kc,, has the values 5870, 85, 0.55, and 0.075 s-l, respectively. The ureasecatalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic
hydrolysis of the following compounds could not be detected: phenyl forrnate, p-nitroforrnanilide, trifluoroacetarnide, p-nitrophenyl carbarnate, thiourea, and 0-rnethylisouroniurn ion.
In theenzyrnatic hydrolysis of urea, the pH dependence of kc,, between pH 3.4 and 7.8 indicates
that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of
methanol gave no detectable methyl carbarnate. A mechanism of action for urease is proposed
which involves initially an 0-bonded complex between urea and an active-site Ni2+ ion and
subsequently an 0-bonded carbamato-enzyme intermediate.
Dixon, N. E., Riddles, P. W., Gazzola, C., Blakeley, R. L. & Zerner, B. (1980) Jack bean urease
(EC 3.5.1.5). V. On the mechanism of action of urease on urea, forrnarnide, acetamide,
N-rnethylurea, and related compounds. Can. J. Biochent. 58, 1335-1 344
Pour la premiere fois, nous montrons que l'acetamide et la N-rnethyluree sont des substrats
de l'urease de la feve Jack. Dans l'hydrolyse enzymatique de l'uree, du forrnarnide, de l'acetamide et de la N-methyluree i pH 7.0 et ii 38"C, les valeurs de kc,, sont respectivement de
5870,'85,0,55et 0,075 s-l. L'hydrolyse de tous ces substrats par l'urease implique le ou les ions
nickel du site actif. Nous n'avons pu deceler l'hydrolyse enzyrnatique des composes suivants:
phenyl forrnate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiouree et
l'ion 0-mCthylisouroniurn. Dans l'hydrolyse enzyrnatique de l'uree, la dependance du kc,, 2
l'egard du pH entre les pH 3,4 et 7,8 montre qu'au rnoins deux forrnes prototropiques sont
actives. L'hydrolyse enzymatique de l'uree en presence de methanol ne donne pas de methyl
carbarnate. Nous proposons un mecanisme d'action pour l'urease. Ce mecanisme irnplique
d'abord la formation d'un complexe entre l'uree et un ion Ni2+du site actif par l'interrnediaire
de liaisons oxygkne. I1 se forme ensuite un intermediaire carbamato-enzyme encore relie par
des liaisons oxygkne.
[Traduit par le journal]
covered that N-hydroxyurea was a substrate ( 4 , 5 ) .
Derivatives of urea which are substrates for urease
Urease is a highly efficientcatalyst for the hydl-olysis
of urea (1, 2 ) , and the specificity of this enzyme was include N-hydroxyurea (4-6), N,N'-dihydroxyurea (7,
believed to be absolute ( 3 ) until Fishbein pr
dis- 8 ) . and ~emicarbazide( 2 ) . he first secure example
of a substrate for urease which is not a substituted
has been
A e s n ~ v ~ a ~ ~ o rEDTA,
us:
ethjlenediaminetetraacetic acid; urea is formamide ( 9 ) . Even though
MES. 2 - ( N - m 0 r ~ h 0 l i ~ ~ ) ~ t
acid;
h ~ ~HEPES,
~ ~ ~ l N-2-hyf ~ ~ i ~ extensively studied for more than 50 years, there has
d r o ~ y e t h y l p i p e r a ~ i n e - . V ' - 2 - ~ t h a ~acid;
~ ~ ~ l fTris,
~ ~ i ~tris(hy- as yet been no reasonable proposition on its mechanism
droxymethy1)aminornethane; kat, katal; A j70. etc., absorbance of action. Because information on substrate specificity
at 570 nrn, etc.
and pH-rate profiles is of critical importance to such
l ~ h i swork was supported in part by the Australian Re- a consideration, we have investigated the action of
search Grants Committee. Correspondence should be ad- urease on a variety of compounds.
Introduction
dressed to Burt Zerner. Some a s ~ e c t sof this work were
presented at an international meeting on "Metal Ion Activation of Biochemical and Chemical Processes" in Canberra,
Australia, in March, 1979.
2Cornmonwealth postgraduate student.
lPresent address; Research School of Chemistry, Australian
National University, Canberra, Australia 2600.
4Revised manuscripts received February 22, 1980.
Materials and methods
Muterials
The ~urificationof urease included ion-exchange chromatography (10, 11). The pH-stat assay of urease has been
described (I, 10). The specific activity of urease (10, 11) was
0008-4018/80/12 1335-10$01 .00/0
@ 1980 National Research Council of Canada/Conseil national de recherches du Canada
CAN. J. BIOCHEM. VOL. 58, 1980
WASTE
\
--a
1
1
f
SOLUTION
A
1
A
a
\
a
PROPORTIONING
-
PUMP
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
MIXING
COIL
FLOW
CELL
-
SOLUTION
B
C
+
O I L
BATH
'(92.C)
\
1
&I
NITROGEN
E
r
L
/
1
WASTE e 2 . 0
SAMPLE
\
L
FIG. 1. Flow diagram for the AutoAnalyzer-based assay system for ammonia. The numbers are nominal flow rates
expressed in millilitres per minute. The compositions of solutions A and B were as follows: (A) 4 L of water, 1 L of
5.75 M acetate buffer (pH 5.51), 5 L of 2-methoxyethanol, and 100 g of ninhydrin; (B) 3.92 L of water, 40 mL of 5.75 M
acetate buffer (pH 5.51), 40 mL of 30'; Brij-35, -0.1 mL of concentrated sulfuric acid, 0.2 g of sodium fluoride, and
1.05 g of hydrazine sulfate.
always between 75 and 93 (mkat/L)/As,o u~lless otherwise ponents (Fig. I). A Cary 14 spectrophotometer equipped with
specified. Protein concentrations were determined spectro- a flow cell (I-cm optical path) was used to measure the abphotometrically (1 1). The concentration of active sites (normal- sorbance at 570 nm produced by the reaction of ammonia with
ity, N) is based on an equivalent weight (1 1) of 96 600 for ninhydrin (Koch-Light, Puriss.) using hydrazine sulfate
.
enzyme of maximal specific activity (93 ( n ~ k a t / L ) ' A ~ , ~ )(British
Drug Houses, AnalaR) as the reducing agent. The
Buffers and pH measurements are described elsewhere (10). system was frequently standardized against solutions of
Methylamine hydrochloride was recrystallized from warm (NH4)2S0,(Schwarz/Mann, ultrapure) in the buffers used for
assays. The dependence ot A570 on ammonium ion concentraethanol, to give mp 226.5-227.4"C, lit. ( 1 2) mp 226-C.
Commercial formamide (BDH, AnalaR. mp 1.5-2.5-C, lit. tion was linear, with a molar absorbance yield (defined as the
(13) mp 2.55"C) was purified by repeated fractional freezing absorbance which would be produced by a solution 1 A4 in
followed by drying over potassium hydroxide at reduced ammonium ion in the sample tube) of 730 M-I cm-I which
pressure t o give mp 1.9-2.6-C. p-Nitroformanilide had mp increased about l o f , over a period of several weeks as the
197.5-198"C, lit. ( 1 4) mp 194-1 9ScC. Acetanlide (Sigma pump tubing aged. Some substrates gave an appreciable stable
Chemical Co.) was recrystallized twice from anhqcirous meth- background absorbance due to slight deconlposition during
anol-ether to giie crystals, n ~ p79.1-81.0°C, lit. (15) nip color development (-7 min at -92-C and pH 5.5).
8 1.6-8 1.8"C. Recrystallized acetamide (3.6 M) in 0.02 M
For rate assays, a solution of 10 mL of substrate in buffer
phosphate buffer (pH 7.1, 1 m h l in EDTA and 5 mhf in was equilibrated at 38-C. A 10-pL aliquot of a solution of
p-n~ercaptoethanol)was treated uith urease (1.6 X lo-' N) dithiothreitol and EDTA was added (to give final concentrafor 7 h at 38°C. After evaporation of water at reduced pres- tions of 2 and 1 pM, respectively) followed by aliquots of
sure, the acetamide was recrystallized to give odorless crystals, buffer or NaF in buffer (50pL) and of enzyme (20-200pL).
mp 79.8-8 1 .O0C. Trifluoroacetamide (Aldrich Chemical Co., The solution was sampled coiltinuously over more than 10 min,
Milwaukee, W1) was recrystallized from water and had mp and initial rates of formation of ammonia were deduced from
the slopes of the recorder traces, The buffers were oxygen-free
74.1-74.8"C, lit. (16) mp 74-75°C.
N-Methylurea(A1drich Chemical Co.) was recrystall~zedfrom 0.05 A2 N-ethylmorpholine.HCl (pH 7.00) and 0.05 M sodium
MES (pH 5.20). Measurements of pH were made at 38'C (21).
chloroform to give needles, nlp 98-99.5'C, lit. (2) mp 100.0100.5"C. After two urease treatments (as above for acetamide) In ever) case the stock e n q me had been dial) zed exhaustively
and recrystallizat ion, it had mp 100.2-1 00.9-C. After three at 4°C against oxygen-free 0.05 M N-ethy lnlorpholine - HCl
crystallizations from nlethanol-ether, 0-methyliso~~ronium butrer (pH 7.00, 1 m M in EDTA and 5 mAl in p-mercaptohydrogen sulfate had mp 118.3-120.5"C, lit. (17) mp 119°C. ethanol).
Thiourea (British Drug Houses) was treated with urease as
above and recrystallized from anhydrous ethanol to give mp HI-dmlysis qf urea
Values of kcat and I(, for the urease-catalyzed hydrolysis of
174.0-175.5"C, lit. (1 8) mp 1814°C. The crystals had the
correct infrared spectrum ( 19). p-Nitrophenyl carbamate had urea at 38°C were determined by initial rate studies at pH
mp 162-164.5"C dec., lit. (20) mp 164-165'C. Methyl car- 3.4-7.8 using a pH-stat (1, 10). Bovine serum albumin (Armour
Pharnlaceutical, fraction V) was dialyzed vs. 0.02 hf phosphate
bamate was purified by vacuum sublimation.
buffer (pH 7.12, 1 m M in EDTA, 5 m M in P-mercaptoethanol),
and then against oxygen-free distilled water. The final assay
Automated cot~tit~irous
analj~sisqf amn~onia
Initial rates of hydrolysis of urea, formamide, acetamide, solution (10.145 mL) contained urea (1-50 mM), dithiotrifluoroacetamide, N-methjlut'ea, thiourea, and O-methyliso- threitol ( I pM), 6-mercaptoethanol (12 pM), EDTA (3 pM),
uronium ion were studied by means of a continuous assay HEPES (5pM), urease (5 X 10-lo N), and bovine serum
M). Before each assay,
system for ammonia based on Technicon AutoAnalyzer com- albumin (0 or, at pH 5 5.2, 1 X
1337
DIXON ET AL.: I1
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
the combination electrode was sequentially soaked at 38°C in
0.1 M HCI (3 min), water (1 min), and 0.02 M phosphate
buffer (pH 7.12, 1 mM in EDTA and 5 mM in /3-mercaptoethanol) for 1 min.
The enzymatic hydrolysis of ['"]urea (The Radiochemical
Centre, Amersham) in the presence of methanol at 38°C was
investigated with the following final concentrations in 10 mL:
[14C]urea (25 mM, 0.026 mCi/mmol; 1 Ci = 37 GBq), phosphate (1 mM), EDTA (55 pM), /3-mercaptoethanol (50 pM),
dithiothreitol (10 pM), methanol (50C; (v/v), -12.7 M), and
urease (0 or 1 X lop6N ) . An apparent pH of 7.0 was maintained by means of a pH-stat for periods between 3 and 40 min
until the total uptake of acid was between -80 and -l00%,
respectively, of the theoretical amount (1). This solution was
added to 250 mL of chloroform which contained 0.5 g of
unlabelled methyl carbamate. The biphasic system was dried
quickly with 50 g of anhydrous CaSO, and the chloroform
was evaporated. The residue was triturated with petroleum
ether (bp 45-65°C). The resulting methyl carbamate crystals
(505; yield) were sublimed it1 vaclio two or three times to constant specific radioactivity (22). Under the conditions of the
[lCC]urea experiments, no enzymatic hydrolysis of 0.1 M
methyl carbamate could be detected, such that k,,,/K,
for
this compound must be less than 0.3 M-I s-l.
Action of rlrease otz phenyl formate attd p-ttitrofornlattilide
The hydrolysis of 1 mkl phenyl formate (23) at pH 7.20 was
monitored spectrophotometrically at 270 nm. The final composition was N-ethylmorpholine buffer (0.049 M), EDTA
(0.13 m M), /3-mercaptoethanol (0.64 mM), acetonitrile (2.125
v/v), potassium phosphoramidate (0 or 20mM), urease
(0 or 1 X
N). For the study of p-nitroformanilide at pH
7.20, the final composition was N-ethylmorpholine.HC1
buffer (0.039 M ) , EDTA (0.16 mM), p-mercaptoethanol (0.82
mM), acetonitrile (2.0C,v/v), urease (2 X
N). At pH
7.2, AE (381 nm) for hydrolysis of p-nitroformanilide was
independently determined to be 12 800 M-I cm-l.
Hydrolysis of'N-rnetliylurea
Initial rates of the enzymatic hydrolysis of N-methylurea
(0.04-0.40 M) were studied at pH 7.00, using the AutoAnalyzer
system to measure IV-methylamine and ammonia. The molar
absorbance yield according to Eq. 1 is 1560 M-I cm-I with
respect to the loss of N-methylurea as determined by calibration with an equimolar solution of ammonia and N-methylamine. Product formation in the presence of urease was linear
with time for at least 30 min in all runs, with hydrolysis of
between 0.02 and 0.16(; of the original N-methylurea. For
product identification, N-methylurea (0.50 M) was treated
with urease (1.0 X lop6N) under precisely the conditions of
the kinetic runs. N-Methylamine was identified by means of a
Technicon TSM amino acid analyzer (24), using N-methylamine hydrochloride as a standard.
Actiotz of lrrease otz p-t~itrophet~yl
carbanlate
The spontaneous decomposition of p-nitrophenql carbamate
was studied spectrophotornetrically at 25.0°C in 0.05 M
phosphate buffers (1.64C; (v/v) in acetonitrile). Yields of
p-nitrophenol were greater than 98';. The ion product of
water (K,) was assumed to be 1 X 10-14. For determination of
the effect of urease, the final composition was MES (0.21 M),
EDTA (0.24 mM), p-mercaptoethanol (1.2 mM), acetonitrile
(2.44C; v/v), p-nitrophenyl carbamate (0.27 mM), and urease
(0 or 2 X
N).
Treatnzetzt of data
Values of kc,, and K,, for enzymatic reactions were obtained
by least-squares analysis of Lineweaver-Burk plots of initial
rates, together with the measured normality of urease. The
calculated percentage uncertainties of kc,, values are twice the
percentage standard error in the intercept of the LineweaverBurk plots, while those for K,, are twice the sum of the percentage standard errors of the slope and intercept.
Results and discussion
Urea
T h e amount of acid o r base required to maintain
the p H constant during the total hydrolysis of 0.25 m M
urea catalyzed by 5 x 10-8 N urease at 38OC was determined by pH-stat. T h e results agree with the theoretical values ( 1) for Eq. 2 within t 1% from p H 3.6
to 8.0 but only within t 5 % at p H 9.5 and 10.1 where
the p H function is more sensitive to small differences
in pH.
Lineweaver-Burk plots for the initial rate of enzymatic hydrolysis of urea were linear in all cases from
p H 3.4 to 7.8, with mean uncertainties of *8% in
k,,, and & 1 2 % in K ,,,. Graphs of k,,, and K,,, as a
function of p H (Fig. 2 ) are not consistent with simple
sigmoid o r bell-shaped profiles. T h e theoretical curve for
the pH-k,.,, profile (Fig. 2 A ) corresponds to Eq. 3 in
[3] SEH,
K ~ ~ ~ . 3 1
-&EN
SEH2
1kl
Products
,'
K
SEH
1k2
Products
~
SE
~
~
which SEH, etc. represent different states of protonation of the enzyme-substrate complex and pKsEH,' =
3.0, P K , , . ; ~ ~=
' 6.25, pKSEH1= 9.0, k1 = 3600 S-l,
and k, = 6350 s-l. The fit of the theoretical curve to
the experimental data is not significantly altered if the
value of pKSmI,' o r pKsEH,' is altered by t O . l o r if
k1 o r k, is altered by t 1 0 0 s-l. T h e k,,, data for the
hydrolysis of urea between p H 3.4 and 7.8 indicate that
the enzyme-substrate complex is active in a t least two
different states of protonation controlled by an apparent pK,' (pKsEH2') of -6.25.
T h e value of 9.0 for pKsEH' in the hydrolysis of urea
( E q . 3 ) was chosen arbitrarily in accord with approximate pK,' values of 8.0-9.0 determined by pHstat and 8.5-9.0 determined in the presence of buffers
( 2 5 ) . This value is also consistent with a pK,' of 9.15
at 25°C for the essential sulfhydryl group of jack bean
urease ( 2 6 ) . While further work is necessary better
to define the effects of p H o n k,,, for urea, the essential aspects of the low p H side of the curve are relatively insensitive to the value of pKSEH'.
'
1338
CAN. J. BIOCHEM. VOL. 58, 1980
1. Urease-catalyzed hydrolysis of urea and of formaTABLE
mide at pH 5.20 and 38°C; competitive inhibition by fluoride
ion
Value
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
Parameter
FIG.2. Effect of pH on the urease-catalyzed hydrolysis of
urea at 38°C. (A) kc,, vs. pH; (B) K , vs. pH. The theoretical
curves correspond to Eq. 3 and Eq. 4 together with the
parameters listed in the text.
The theoretical curve for the pH-K, profile (Fig.
2B) corresponds to Eq. 4 where H,E etc. represent
different states of protonation of the free enzyme, and
pKHtE' = 2.0, pKHEf= 6.5, K, = 100 mM, K, = 1.9
rnM, and K, = 3.3 mM. Because of the lesser reliability of the data at very low pH, the values of K , and
pKHgE'are only approximate. It is clear, however, that
K, increases markedly at pH values less than 4, and
appropriate control experiments establish that this effect is not due to chloride ion. It is similarly clear that
K,, for urea has a moderate dependence on an apparent
pK,' of -6.5.
The value of lo-, k,,, for urease at pH 7.00 is 5.87,
4.47, 3.69, and 3.01 s-1 at 38.0°C, 29.8, 24.8, and
19.8, respectively, as determined by pH-stat. The value
of K,, (2.9 m M ) is independent of temperature ( 2 7 ) .
Lineweaver-Burk plots for the urease-catalyzed
hydrolysis of urea at pH 5.20 according to Eq. 2 in
Ureaa
Formamideb
aStudied with the AutoAnalyzer system in the presence of 0, 2, or 4 pM
sodium fluoride. [Ureaselo = 8.0 X 10-'0 N. [Urealo = 1.2-25 mM.
b[~rease]o= 4.0 X
N. [Sodium fluoride] = 0, 2.4, 4.8, or 9.7 pM.
[Formamide]~= 0.1-1.2 M.
CMeasured in a completely independent experiment.
the absence and presence of fluoride ion are linear,
and the values of kc,, and K,, are listed in Table 1.
These values were obtained in the presence of 0.05 M
MES buffer using the AutoAnalyzer assay system, and
they agree within experimental error with those determined by pH-stat (Fig. 2 ) , thus indicating the absence of specific buffer effects. Fluoride ion behaves
as a simple competitive inhibitor of the urease-catalyzed hydrolysis of urea at pH 5.20 and 38"C, with a
K i of 7.0 + 1.0 pM (Table 1) .
The enzymatic hydrolysis of [14C]urea was carried
out at 38°C in the presence of methanol at p H 7.0 in
order to see if a carbamoyl-enzyme or carbamatoenzyme intermediate could be trapped to form methyl
carbamate. In a series of experiments, radioactivity
corresponding to 0.01 to 0.02% of the initial amount
of [14C]urea was found in association with added, unlabelled methyl carbamate, irrespective of the presence
or absence of urease. Control experiments established
that if formed, methyl [lT]carbamate would not have
been enzymatically destroyed. These results provide
absolutely no evidence for the formation of methyl carbamate during the enzymatic hydrolysis of urea in the
presence of methanol. A maximum value of less than
may be calculated for k,,,oII/kH,o where the rate
constants refer to attack of methanol or water on a
putative carbamoyl-enzyme or carbamato-enzyme intermediate.
Formamine
The initial rate of hydrolysis of 0.75 M formamide at
pH 7.00 and 38°C is directly proportional to the concentration of urease (0.3 x 10-7 - 1.4 x 10-7 N ) ,
using the AutoAnalyzer system to measure the rate
of formation of ammonia. Lineweaver-Burk plots for
the enzymatic hydrolysis of formamide are linear, and
the Michaelis-Menten parameters are listed in Table 2.
Fluoride ion has no effect on the maximum rate, and
DIXON ET AL.: I1
1339
TABLE
2. Urease-catalyzed hydrolysis of formamide, acetamide
and N-methylurea at pH 7.00 and 38°C; competitive inhibition
by fluoride ion
Value
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
Parameter
Formamidea Acetamideb N-MethylureaC
a[Urease]o= 1.21 X lo-' N. [Sodium fluoride] = 0.0.24, 0.5, or 1.0 m 'M.
bconditions are described in Fig. 3.
CConditions are described in Fig. 4.
dMeasured in a completely independent experiment.
a graph of the slopes of the Lineweaver-Burk plots vs.
[fluoride] is linear (27, 28). The value of K iis 1.32 -C
0.35 m M for fluoride ion acting as a competitive inhibitor of the urease-catalyzed hydrolysis of formamide
at p H 7.00 and 38°C (Table 2).
Fluoride ion also behaves as a simple competitive
inhibitor of the urease-catalyzed hydrolysis of formamide at p H 5.20 (27, 2 8 ) . The K iof fluoride ion at
p H 5.20 and 3 8 " C is 8.2 L 1.7 r M (Table 1 ) .
Pherlyl fortnate and p-nitrofornznnilide
The hydrolysis of phenyl formate at p H 7.20 and
25°C followed first-order kinetics. The value of k,,,,
(2.17 x 10-%-1)
in the presence of 1 x l o - > N
urease was 3 0 % higher than in its absence. However,
the value in the presence of urease was unaltered when
the enzyme had previously been 99.8% inhibited by
reaction with 20 m M potassium phosphoramidate ( 2 9 ) .
The urease-promoted hydrolysis of phenyl formate
therefore does not involve the active site and is presumably analogous to the hydrolysis of o-nitrophenyl
oxalate anion catalyzed by bovine ribonuclease A (30)
and the decomposition of 1,l -dihydro-2,4,6-trinitrocyclohexadienate ion catalyzed by bovine serum
albumin ( 3 1) .
N o hydrolysis of 0.2 m M p-nitroformanilide was detected in 1.5 h at 25°C in the presence of 2.0 X lo-;'
N urease at p H 7.20.
A ce tamide
Urease catalyzes the hydrolysis of acetamide at p H
7.00 to form ammonia, as assayed by the AutoAnalyzer
system. Product formation was linear with time for at
least 3 0 min in all runs, with hydrolysis of between
0.04 and 0.18% of the original acetamide (0.11-1.3
M ) . The initial rate of the enzymatic formation of
ammonia is directly proportional to the concentration
of urease (0.4 x 10-6 - 4.4 X 1O-G N ) .
Initial rates of the urease-catalyzed hydrolysis of
acetarnide obey Michaelis-Menten kinetics as shown
FIG.3. Inhi bition of the urease-catalyzed hydrolysis of
acetamide by fluoride ion at pH 7.00 and 38°C. (A) Lineweaver-Burk plots in the presence of 0 (C),
0.63 mM (O),
1.26 mkl ( A ) ,and 2.53 mM (0)
sodium fluoride. [Urease]~=
8.91 X lo-' N. Each least-squares line is based on the four
points of highest substrate concentration. The filled circles (e)
refer to urease of specific activity 30C, less than that used for
the other runs. (B) Plot of the slopes from A vs. [fluoride].
by a Lineweaver-Burk plot (Fig. 3A, Table 2 ) . he
same plot also contains points determined with urease
of lower specific activity. Their good fit to the line
establishes that the portion of urease which is inactive
toward urea (as judged by a low specific activity) is
also inactive toward acetamide. It should be noted that
when the acetamide concentration is less than -0.2 M,
the observed initial rate is greater than that predicted
from the Michaelis-Menten parameters in Table 2.
It is possible that the high concentrations of acetamide
are producing as yet undefined substrate activation o r
medium effects.
Lineweaver-Burk plots for the urease-catalyzed
hydrolysis of acetamide in the presence of fluoride ion
are also shown in Fig. 3A. A graph of the slopes of
the lines in Fig. 3A vs. [fluoride] is linear (Fig. 3B),
leading to the value of 2.21 & 0.64 m M for K i of
fluoride ion competing with acetamide at p H 7.00 and
38°C (Table 2 ) . The decreased maximum velocity in
1340
CAN. J. BIOCHEM. VOL. 58, 1980
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
not significantly different. It may be estimated that the
maximum possible value of k,,,/K, for trifluoroacetamide would be 1 0 . 0 4 M-I s-' (on the assumption that K,,, >>20 m M ) .
FIG.4. Inhibition of the urease-catalyzed hydrolysis of
N-methylurea by fluoride ion at pH 7.00 and 38°C. (A) Lineweaver-Burk plots with [ureaseIo= 2.75 X
N in the
0.63 mM (m), 1.26 mll.1 ( A ) , and 1.90 mM
presence of 0 ((I),
( V ) sodium fluoride. The filled circles (e)refer to N-methylurea which had been subjected to only one cycle of urease
treatment - recrystallization. (B) Plot of the slopes from A vs.
[fluoride].
the presence of fluoride ion is qualitatively consistent
with a ternary acetamide-fluoride-urease complex,
analogous to the urea-fluoride-urease
complex previously observed at pH 7.0 (29). The deviations from
Michaelis-Menten kinetics at low substrate conccntrations also occur in the presence of fluoride ion (Fig.
3 A ) . All of these findings clearly indicate that the
kinetics of the urease-catalyzed hydrolysis of acetamide
at pH 7.00 are as yet imperfectly defined. Nevertheless
this work constitutes the first demonstration that
acetamide is a substrate for urease (cf. Ref. 9 ) .
Trifluoroacetanzide
The initial rate of spontaneous hydrolysis of 20 m M
trifluoroacetamide at pH 7.00 and 38°C was 0.14 p M
s-l as studied with the AutoAnalyzer system. In the
presence of 1.8 x 10-6 N urease, the initial rate was
N-Methylurea
The enzymatic hydrolysis of 0.5 M N-methylurea
was carried out at pH 7.00 until the extent of hydrolysis
was 1.13% (after 3 h ) as calculated from the
Michaelis-Menten parameters in Table 2. When the
solution was analyzed by means of an amino acid
analyzer, the N-methylamine peak was well resolved
from the ammonia peak and corresponded to 98.7%
of the theoretical value. When the extent of hydrolysis
was calculated to be 2.25% (after 6 h ) , the apparent
yield of N-methylamine was somewhat less (88.8% of
the theoretical value) because of inadequate resolution
of the ammonia and methylamine peaks. A control
reaction mixture showed that methylamine was not
formed in the absence of urease. These results validate
the stoichiometry of Eq. 1.
The initial rate of hydrolysis of 0.40 M N-methylurea
at pH 7.00 is directly proportional to the concentration of urease (0.7 x 10-6 - 2.8 x 10-W) . Initial
rates of the urease-catalyzed reaction obey MichaelisMenten kinetics as shown by a Lineweaver-Burk plot
(Fig. 4 A ) . The same plot also shows that omission of
one cycle of pretreatment of the N-methylurea with
urease does not alter its susceptibility to urease. Values
of k,.,, and K,, are given in Table 2.
Lineweaver-Burk plots for the urease-catalyzed
hydrolysis of N-methylurea at pH 7.00 in the presence
of fluoride ion are also shown in Fig. 4A. From Fig.
4B, the value 2.24 t 0.56 m M is obtained for the apparent dissociation constant K , of fluoride ion competing with N-methylurea at p H 7.00 (Table 2 ) . The
decrease in maximum velocity in the presence of
lluoride ion would be explained if a ternary Nmethylurea-fluoride-urease complex, analogous to the
urea-fluoride-urease complex ( 2 9 ) , were formed.
These results establish for the first time that Nmethylurea is a substrate for urease, since a previous
report to that effect (25) has been shown to be in error
by several orders of magnitude ( 2 ) .
p-Nitrophenyl carbat?iate
The spontaneous formation of p-nitrophenol from
p-nitrophenyl carbamate in phosphate buffers at pH
values between 6.0 and 7.5 obeys a first-order rate law
for at least three half-lives. A graph of k,,,, vs. hydroxide ion concentration is strictly linear giving a value
for k,,- of 2.64 x 10-3 M-1 s-1 at 25"C, consistently
with hydroxide-promoted formation of p-nitrophenoxide ion and cyanic acid (32, 3 3 ) .
The effect of urease on the rate of decomposition
of p-nitrophenyl carbamate was studied in 0.21 M NlES
buffer in order to avoid inhibition by phosphate
monoanion (29). The formation of p-nitrophenol obeys
a first-order rate law for more than three half-lives
1341
DIXON ET AL.: I1
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
under all conditions. The observed rate constant is
(2.52 k 0.01) X 10-G-I at pH 6.03, independently
N urease. These experiof the presence of 2 x
ments provide no evidence whatsoever for urease
catalysis of the hydrolysis of p-nitrophenyl carbamate.
It may be estimated that the maximum possible value
of k,,,/K,, for this compound would be less than 0.4
M-I s-I (on the asrumption that K,, >> 0.27 m M ) .
Bennett and Wren (34) report that at pH 6.0 and
2S°C, p-nitrophenyl carbamate does not undergo spontaneous hydrolysis but is a good substrate for urease.
having the same maximum velocity (k,.,,) as urea and
a K,, of 0.67 mM. Unfortunately, they give insufficient
details to allow a critical assessment of their experiments.
Thiourea
N urease on 1 M thiourea
The effect of 2 x
was studied at pH 5.2 and 38°C (with and without
5 X 10-5 M sodium fluoride) and at pH 7.0 by means
of the AutoAnalyzer system. At the limit of sensitivity
of the technique, no evidence of enzymatic hydrolysis
of thiourea could be obtained. At both pH 7.0 and 5.2,
the value of k,,,, can be calculated to be 5 1 X lo-:: s-'
(or, if K,,, is much greater than 1 M, k,.,,/K,,, would be
< 1 x 1 0 - W - I s-I). In contrast, Bennett and Wren
(34) reported that at pH 6.0 and 2S°C, thiourea is a
substrate for urease and has a K,,, of 0.21 M and a
maximum velocity (k,,,,) identical to that of urea.
Their experimental details are insufficient to allow
repetition of their work, and we can offer no positive
explanation of their reported results.
ion
0-Methy lisouror~i~rrn
There was no detectable hydrolysis of 23 mM Omethylisouronium ion at 38°C and pH 7.0 in the
presence or absence of 5 x lo-'; N urease as studied
by the AutoAnalyzer system. From the limits of sensitivity of the experiments, the maximum possible value
of k,.,,/K,, for 0-methylisouronium ion would be 0.03
M-I s-l. The pK,,' of this ion is 9.72 at 24°C ( 3 5 ) , so
that it is predominantly in the positively charged form
under these conditions.
A corninon active site for the hydrolysis of urea,
for111ainidc,acctainidc, and N-incthyl~rrea
Fluoride ion binds to the tightly bound nickel ion(s)
in urease with a dissociation constant of 1.23 ? 0.10
mM at pH 7.12 and 2S°C, as determined spectrophotometrically by competition with P-mercaptoethanol
(29). The dissociation constant of the fluoride-urease
complex has also been measured at pH 7.00 and 38°C
by competition with several substrates. The values so
determined are 1.O1 k 0.08 mM (urea (29) ), 1.32 k
0.35 rnM (formamide, Table 2 ) , 2.21 ? 0.64 m M
(acetamide, Table 2 ) , and 2.24 ? 0.56 mM (Nobtained with
2). The
acetamide and N-methylurea as substrates appear to be
slightly higher than those derived from urea and formamide : the results with acetamide are complicated by
the ill-defined substrate activation effects mentioned
earlier, and the high concentrations of urease in the
N-methylurea experiments led to relatively noisy
recorder traces because of some precipitation in the
AutoAnalyzer heating bath. It is therefore likely that
all these kinetically determined values of the dissociation constant of the fluoride-urease complex are
identical to each other (within the combined uncertainties of the systems) and to the spectrophotometrically determined dissociation constant. Further, the
dissociation constant of the fluoride-urease complex
at p H 5.20 and 38°C is -7 P M (Table 1 ) regardless
of whether urea or formamide is the substrate. These
facts establish that the various substrates are hydrolyzed at the same site, and that this site involves the
tightly bound nickel ion(s) of urease:;
Substrate spc~cificityof urcase
The efJcct of p H on k,.,, and K,, for different
s~ibstrates
The maximum value of kc,, for the urease-catalyzed
hydrolysis of urea at 38°C occurs at pH -7.4, and the
T, 2 / (k,.nt)pIT
i , O is 0.68. While detailed
ratio
pH-k,.,, profiles are not yet available for the hydrolysis
5.2/ (k(.at)gH
7.0
of other substrates, the ratio
for formamide is 2.4. For semicarbazide, the ratio
(k,.,t),11 o l (ktnt)l,H7 . 0
1.70 at 38°C (Table 3 ) .
Thus, the pH-kc,, profiles for formamide and semicarbazide must be markedly different from that of urea.
,
Str~rcture-reactivity relationships \rlith lirease substrates
With the presently available examples, alteration of
the structure of a substrate for urease in a manner
which increases the rate of its hydroxide-promoted
hydrolysis or decomposition generally results in a decrease in efficiency of its urease-catalyzed hydrolysis.
In the formate series, formamide has a k,,,,/Kn1 of
-80 M-I s-I at pH 7.00 and 38°C while no enzymatic
reaction with either phenyl formate or p-nitroformanilide could be detected. In the acetate series,
acetamide has a k,,,/K,, of 0.73 M-I s-I at pH 7.00
and 38"C, while no detectable enzymatic hydrolysis
of trifluoroacetamide occurred. In the urea series, the
k,,,,/K,,, for urea itself is 2.0 x 10"-I
s-I at pH 7.00
and 38°C while enzymatic hydrolysis of the related
esters, methyl, ethyl, and p-nitrophenyl carbamate, was
not detected ( 2 ) . Further, all substituted ureas currently known to be substrates (N-hydroxy- (4-6),
N,N'-dihydroxy- (7, 8 ) , N-amino- (semicarbazide,
Table 3 ) , and N-methylurea (Table 2 ) ) have markedly
lower k,,, and k,.,,/K,,, values than urea itself, in spite
of the fact that the inductive effect of the substituent
varies appreciably.
TJrease appears to catalyze the breakdown of iV-nitrourea
in the presence of 9 % (v/v) ethanol at pH 5.0 and 38°C. The
M-l s-l. While this
nominal value of kc,,lK,
is I
presumably occurs at the active site, studies on the effect of
fluoride have not yet been undertaken.
1342
CAN. J. BIOCHEM. VOL. 58, 1980
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
In contrast to these results with urease, the rates
of reaction of carboxylic acid derivatives with the nonmetalloenzymes, a-chymotrypsin ( 3 6 ) , and the liver
carboxylesterases (37, 38) roughly parallel their susceptibility to nucleophilic attack by hydroxide ion. The
differences in structure-reactivity relationships between
urease and the serine proteinases and esterases are consistent with a key mechanistic role for the Lewis acid,
Ni", in the mechanism of action of urease.
An exception to the general structure-reactivity relationship of urea analogs with urease is the fact that
k,,, for formamide is 160 times that for acetamide at
pH 7.0. For comparison, the rate constant for alkaline
hydrolysis of formamide is 46 times that for acetamide
(39, 40). This exception would be explained by an
unfavorable steric interaction of acetamide with urease.
The same effect would account for the very low efficiency of enzymatic hydrolysis of N-methylurea as
compared with urea at pH 7.0.
A nzechnnist?l of rrctiolt for urense
Urease is an extraordinarily efficient catalyst of the
/zydrolysis of urea, the uncatalyzed reaction being undetectably slow. Since this is the only known nickel
metalloenzyme and since it contains 2 g-at. of nickel
per mole of active sites, we speculate at this stage that
the catalytic activity, specificity, and "unusual" chemistry of urease may all derive from a mechanism which
involves both of the nickel ions. The proposed structure of the resting enzymc at neutral pH contains a
water molecule coordinated to one of the nickel ions
and an hydroxide ion coordinated to the other (Scheme
1 ) . The four stages of the mechanism are as follows.
Tan1 L 3. Urease-catalyzed hydroljsis of seniicarbazide at
38 C n
Value
Parameter
p H 5.00
p H 7.00
aRecalculated from previously published data (2).
(i) The substrate is activated toward nucleophilic
attack by 0-coordination to a Ni2+ ion, by analogy
with the activation of dimethylformamide ( D M F ) in
[Co(NH,) ,DMFI3+ toward attack by hydroxide ion
+
(41). The =NH, of the coordinated substrate interacts with a nearby negatively charged group. Because
urease is irreversibly inhibited by triethyloxonium ion
( 4 2 ) , we postulate that this group is a carboxylate ion.
(ii) A nickel-coordinated hydroxide ion attacks the
carbonyl carbon of the coordinated substrate to form
a tetrahedral intermediate. This finds analogy in the
facile intramolecular hydrolysis of the carboxylic acid
amide bond in [(en),Co(OH) (GA)]", where en is
ethylenediamine and GA is glycine amide coordinated
to Co(ll1) through its a-amino group (43). Similarly,
hydroxide ion coordinated to Ni(I1) or Co(I1) is a
very efficient nucleophile in the intramolccularly
catalyzed hydrolysis of esters (44).
(iii) Thc breakdown of the tetrahedral intermediate
to form a coordinated carbamate or carboxylate ion is
facilitated by the active-site sulfhydryl group (26, 27)
acting as a general acid catalyst.
(i\>)Replacement of the coordinated carbamate ion
or carboxylate ion by water leads to regeneration of
the enzyme. Carbamate ion is known to be the product
of the action of urease on urea ( 6 ) .
Methanol does not trap any intermediate in either
the urease-catalyzed hydrolysis of urea or the bovine
carboxypeptidase A catalyzed hydrolysis of substrates
(45). Breslow has interpreted the latter result recently
in terms of 0-coordination of the substrates of carboxypeptidase A to the active-site zinc ion (45) and
previously in terms of a zinc-bound hydroxide ion acting as a nucleophile (46).
Two limiting resonance structures (1 and 2)
may be drawn for an 0-bonded complex of a sub-NHNH,,
strate with urease (where -R = -NH,,
-NHOH, -NHCH3, -H, o r -CH3). The pK,' for
loss of a proton from the substrate in the complex at
the active site will be determined by the degree of
DIXON ET AL.: I1
charge separation in the complex (i.e., 1 vs. 2 ) . For
= -NH,)
would be -14
example, the pK,' of 1 (-R
since 1 resembles the free substrate (urea ( 4 7 ) ) while
that of 2 would be appreciably less than 9.7 (by
extrapolation from 0-methylisouronium ion (35) by
allowance for the inductive effect of a metal ion in
place of a methyl group). A greater charge separation
(i.e., a greater resonance contribution of 2 as compared with 1 ) corresponds to a lower pK,' for the sub-
1343
series of papers and in earlier papers from this laboratory, as well as those of other students whose work
may not have been specifically cited.
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
1. Blakeley, R. L., Webb, E. C. & Zerner, B. (1969) Biocl~ernistry8, 1984- 1990
2. Gazzola, C., Blakeley, R. L. & Zerner, B. (1973) Can.
1.Bioclzem. 51, 1325-1330
3. Varner, J. E. (1960) in The Enzymes (Boyer, P. D.,
C
Lardy, H. & Myrback, K., eds.), 2nd ed., vol. 4, pp.
strate =NH, of the nickel-substrate complex a t the
247-256, Academic Press, New York, NY
active site. Nucleophilic attack is favored by resonance
4. Fishbein, W. N., Winter, T. S. & Davidson, J. D. (1965)
form 2 of the coordinated substrate (Scheme 1 ) ; but
J. Biol. Clzem. 240, 2402-2406
if the pK,' were too low, then enzymatic activity would
5. Fishbein, W. N. & Carbone, P. P. ( 1965) 1.Biol. Chern.
be negligible at neutral p H due to dissociation of a
240,2407-24 14
However, the postulated carproton from 1-2.
6. Blakeley, R. L., Hinds, J. A., Kunze, H. E., Webb, E. C.
& Zerner, B. ( 1969) Bioclzer?zistry8, 199 1-2000
boxylate anion suitably positioned very close to the
7. Fishbein, W. N. (1968) Anal. Clzinz. Acta 40, 269-275
amide nitrogen atom in the complex (Scheme 1)
8. Fishbein, W. N. ( 1969) J. Biol. Cllern. 244, 1188-1 193
would both increase the contribution of 2 relative to 1
9. Fishbein, W. N. (1977) Bioclzir?l. Biophys. Acta 484,
and raise the pK,' of the coordinated substrate. The
433-442
unfavorable steric interaction of N-methylurea and 10. Dixon, N. E., Gazzola, C., Asher, C. J., Lee, D. S. W.,
acetamide with the active site of urease (vide s u p r a )
Blakeley, R. L. & Zerner, B. (1980) Can. J. Biochem.
could cause an unfavorable positioning of the amide
58,474-480
nitrogen vis-a-vis the carboxylate ion and hence ac- 11. Dixon, N. E., Hinds, J. A., Fihelly, A. K., Gazzola, C.,
Winzor, D. J.. Blakeley, R. L. & Zerner. B. (1980)
count for their very low kc,, values at p H 7.0.
Can. J. Bioclzer,i. 58, 1323-1334
The different pH-k,,,
profiles for some different
substrates of urease can be understood in terms of 12. Astun, J. G. & Ziemer, C. W. (1946) 1. Arn. Chenz.
SOC.68. 1405-1413
effects of substrate structure on the pK,' of the Ni2+13. Smith, G. F. ( 1931) J. Chern. Soc. 3257-3263
coordinated substrate, independently of interactions 14. De Wolfe, R. H. & Newcomb, R. C. (1971) J. Org.
with the nearby carboxylate ion. Thus the lower p H
Chern. 36, 3870-3878
optimum for formamide as compared with urea is con- 15. Davies, M., Jones, A. H. & Thomas, G. H. (1959)
sistent with the lower pK,' of simple imidoesters (e.g.,
Trans. Far-aday Soc. 55, 1100- 1108
+
16. Corley, R. S., Cohen, S. G., Simon, M. S. & WolosinH,C-C(=NH,)OC,H,,
pK,' 7.50 at 2S°C, and p =
ski, H. T. (1956) J. A m . Chern. Soc. 78, 2608-2610
0.5 ( 4 8 ) ) as compared with 0-methylisouronium ion 17 Fearing, R. B. & Fox, S. W. (1954) J. A m . Clzem. Soc.
(pK,' 9.72 (35) ) . Extension of this argument would
76,4382-4385
account for the lack of detectable enzymatic hydrolysis 18. Shnidman, L. (1933) 1. Plzys. Chern. 37, 693-700
of trifluoroacetamide as contrasted with the nearly 19. Stewart, J. E. (1957) 1. Chem. Phys. 26, 248-254
20. Robillard, G. T., Powers, J. C. & Wilcox, P. E. (1972)
isosteric substrate acetamide at p H 7.0.
Bioclzenlistry 11. 1773-1784
The detailed mechanism of Scheme 1 requires that
21. Bates, R. G. (1964) Determirzation o f pH. Tlzeory arzd
the two nickel ions per 96 600-dalton subunit of urease,
Pracvice, Wiley, New York, NY
be within -6 A ( 1 A = 0.1 nm) of each other. How- 22. Dixon, N. E., Gazzola, C., Watters, J. J., Blakeley, R.
ever, the essential elements of the mechanism could
L. & Zerner, B. (1975) 1. A m . Chem. Soc. 97, 4130remain unaltered even if this proves to be incorrect.
4131
We note that as yet there are no models of activation 23. Stoops, J. K., Horgan, D. J., Runnegar, M. T. C., de
Jersey, J., Webb, E. C. & Zerner, B. (1969) Bioof carboxylic acid amides (as differentiated from carchenzistry 8, 2026-2033
boxylic acid esters ( 4 4 ) ) towards hydrolysis by Ni2+
ion, and, in so far as we have been able to determine, 24. Scott, K. & Zerner, B. (1975) Can. J. Bioclzern. 53,
561-564
no models at all for metal ion promoted hydrolysis of 25. Sundaram, P. V. & Laidler, K. J. (1970) Can. J. Biourea. Because of the absence of such data we have
chern. 48, 1132-1 140
been forced to model the enzyme on itself.
26. Norris, R. & Brocklehurst, K. (1976) Biochern. 1. 159,
Future work from this laboratory will be concerned
245-257
to test consequences of the mechanism here proposed 27. Dixon, N. E. (1978) Ph.D. thesis, University of Queensin an attempt further to delineate the detailed mechaland, Brisbane, Australia
nism of action of this enzyme.
25. Riddles, P. W. (1980) Ph.D. thesis, University of
Queensland, Brisbane, Australia
29. Dixon, N. E., Blakeley, R. L. & Zerner, B. (1980)
Acknowledgement
Carz. 1.Biochenz. 58, 481-488
One of us (B.Z.) would like to acknowledge the 30. Bruice, T. C., Holmquist, B. & Stein, T. P. (1967) J.
contributions of all the other authors listed in this
A m . Chern. Soc. 89,4221-4222
1344
CAN. J. BIOCHEM. VOL. 58, 1980
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by San Diego (UCSD) on 04/19/17
For personal use only.
31. Taylor, R. P., Chau, V., Bryner, C. & Berga, S. (1975)
J. Am. Chem. Soc. 97, 1934-1943
32. Dittert, L. W. & Higuchi, T. (1963) J. Pharm. Sci. 52,
852-857
33. Bender, M. L. & Homer, R. B. (1965) J. Org. Chem.
30,3975-3978
34. Bennett, J. & Wren, E. A. (1977) Biochim. Biopllys.
A cra 482,42 1-426
35. Zief, M. & Edsall, J. T. (1937) J. Arn. Chem. Soc. 59,
2245-2248
36. Zerner, B., Bond, R. P. M. & Bender, M . L. (1964) J.
Am. Clzem. Soc. 86,3674-3679
37. Stoops, J. K., Horgan, D. J., Runnegar, M. T. C., de
Jersey, J., Webb, E. C. & Zerner, B. (1969) Bioclzernisrry 8, 2026-2033
38. Stoops, J. K., Hamilton, S. E. & Zerner, B. (1975)
Can. J. Bioclzem. 53, 565-573
39. Jencks, W. P. & Gilchrist, M. (1964) J. Arn. Chern.
SOC.86, 5016-5020
40. Bolton, P. D. & Jackson, G. L. ( 1971) Arrsr. J. Chem.
24,969-974
41. Buckingham, D. A., Harrowfield, J. MacB. & Sargeson,
A. M. (1974) J. Am. Chem. Soc. 96, 1726-1729
42. Gazzola, C., Blakeley, R. L. & Zerner, B. (1972) Proc.
A~tst.Biochem. Soc. 5, 1 1
43. Buckingham, D. A., Keene, F. R. & Sargeson, A. M.
(1974) J. Am. Chem. Soc. 96,4981-4983
44. Wells, M. A. & Bruice, T. C. (1977) J. Am. Chem.
SOC.99,5341-5356
45. Breslow, R. & Wernick, D. L. (1977) Proc. Narl. Acad.
Sci. U.S.A. 74, 1303-1307
46. Breslow, R., McClure, D. E., Brown, R. S. & Eisenach,
J. (1975) J. Am. Clzern. Soc. 97, 194-195
47. Woolley, E. M. & Hepler, L. G. (1974) Anal. Chem.
44, 1520-1523
48. Pletcher, T. C., Koehler, S. & Cordes, E. H. (1968)
J. Am. Chem. Soc. 90,7072-7076