t r ( ) ( ) R (ri . , ,
. , r , , n r r sr n y 1 7 . 4 0 0 - 4 0 9 ( 1 9 8 9 )
LactateDehydrogenase-Catalyzed
Regenerationof NAD from
NADHfor Use in Enzyme-Catalyzed
Synthesis
H . Kr,trH C s EN n u l r A N D Geoncs M. W S TTE S TD E S
Department of Chemistrt', Huruard Uniuersity, Cambridge, Massachusetts 02138
ReceiuedOctober 24, 1988
L a c t a t e d e h y d r o g e n a s e( L D H , r a b b i t m u s c l e . E C 1 . 1 . 1 . 2 7 ) c, o u p l e d w i t h p y r u v i c a n d
gfyoxylic acids as oxidants, regenerates NAD in .situ for enzyme-catalyzed oxidations. The
use of LDH has advantages of convenience, stability of the regenerating system, and economy. Pyruvate and glyoxylate differ in their advantages and disadvantages as oxidants.
Pyruvate analogs, monomethyl oxalate and S-methyl thiooxalate, were examined as possipress,Inc.
ble oxidants but were not substrates of lactate dehydrogenase. o rg8gAcademic
INTRODUCTION
W e her ein d e s c ri b e th e u s e o f l -l a c ta te dehydrogenase(LD H .r rabbi t muscl e,
E C L l . 1 . 2 7 ) . w i t h p y r u v i c a n d g l y ' o r y ' l i ca c i d sa s o x i d a n t s .t o r e g e n e r a t e
NAD for
e n z y m e - c a t a l y ' z ep
d r. e p a r a t i v eo x i d a t i o n s .N A D r e g e n e r a t i o n
u s i n gL D H h a s a d v a n t a g e so f c o n v e n i e n c e .s t a b i l i t yo' f t h e r e g e n e r a t i n g
, nd
e n z y ' m ea n d r e a g e n t s a
e c o n o m ) ' .I n s y ' s t e m sn o t s e n s i t i v et o d e a c t i v a t i o nb y g l y o x y l a t e .t h e h i g h r e d o x
pot ent ial of g l y o x y l a te i s a n a d v a n ta g e .
Enzymes are useful as catalysts for organic synthesis (1), and methods for
cofactor regenerationhave made synthetic applicationsof even cofactor-requiring
enzvmes practical (2). Although several good to excellent systems regeneratethe
reduced nicotinamide cofactors, NAD(P)H (2, 3), methods for regeneratingthe
oxidized cofactors. NAD(P), are less developed and are currently being investigated (4-7). A frequent problem of enzyme-catalyzedoxidations is not the cofactor-regeneratingstep but the oxidation of substrate in the synthetic step. Biochemical oxidations may suffer from unfavorable thermodynamics and
noncompetitive and uncompetitive product inhibition (4, 8).
LDH catalyzes the completely stereoselectivereduction of pyruvic acid (with
c o n c o m i t a n to x i d a t i o no f N A D H ) t o r , - l a c t i ca r c i d E
, q. il1, R : CHr e, l0). LDH
als o ac c ept s a s s u b s tra te sa v a ri e ty o f o ther a-oxo aci ds (l l , l 2), i ncl udi ng gl yo x y l i c a c i d , w h i c h i s r e d u c e di n t h e p r e s e n c eo f N A D H t o g l y c o l i ca c i d . E q . [ ] , R
' Abbreviations
used:FMN, flavinmononucleotide;
G6P.glucose
6-phosphate;
G6PDH,glucose-6phosphate
dehydrogenase;
GluDH,glutamate
dehydrogenase;
Gly-Gly,glycylglycine;
Hepes,4-(2hydroxyethyl-l-piperazineethanesulfonic acid; HLADH, horse liver alcohol dehydrogenase; aKG,
ammonium a-ketoglutarate; LDH, L-lactate dehydrogenase; Mops, 4-morpholinepropanesulfonic
acid: TEA. triethanolamine.
0045-2068/89
$3.00
C o p y r i g h t O 1 9 8 9b y A c a d e m i c P r e s s , I n c .
All rights of reproduction in any form reserved.
LACTATE DEHYDROGENASE-C
AT ALY 7.EDREGENERATION
401
: H ( 1 J - 1 5 ) . T h e u t i l i t y o f L D H i n p r e p a r a t i v er e d u c t i o n s i. t s e a s eo f i m m o b i l i z a t i o n a n d m a n i p u l a t i o n .a n d i t s s t a b i l i t y w h e n p r o t e c t e df r o m a u t o x i d a t i o nh a v e
( 1d2 , I 6 \ .
beendemc-rnstrate
NADH
NAD
ul
n\/
8\?'
R^coo-
LDH
R= cHs,H
n^coo-
We have examined LDH as a catalyst for NAD regeneration. The two most
commonly reported methods for regeneratingNAD(P) use ammonium a-ketoglut a r a t e ( a K G ) a n d g l u t a m a t ed e h y d r o g e n a s e( G l u D H , E C L 4 . 1 . 3 ) ( 4 , 8 , 1 7 ) o r
F M N / O r . w i t h ( 6 ) o r w i t h o u t ( 4 , 1 8 ,1 9 ) F M N r e d u c t a s e( E C 1 . 6 . 8 1. ) a s a c a t a l y s t .
L D H is les s ex p e n s i v ea n d h i g h e r i n s p e c i fi cacti vi ty than both GIuD H and FMN
r e d u c t u s e . rU n l i k e F M N i o r . L D H p e r n - r i t sN A I ) r e g e n e r e r t i ounn d e r a n a e r o b i c
co ndit ions ( m an y e n z y me s a re d e a c ti v a te db1,di or,r-gen.superoxi de.and peroxi d e) . P y r uv ic an d g l y o x y l i c a c i d s a re l e s s e rp ensi ve than a-ketogl utari caci d and
F M N , 3 a n d g l y o x y l a t e( E o : - 0 . 9 0 V a ) i s m o r e s t r o n - e l o
y x i d i z i n gt h a n a - K G ( E ;
- - 0. l2l V f or re d u c ti v e a m i n a ti o n ).
P ot ent ialobs ta c l e sto th e u s e o f p y ru v a te a s the ori dant w ere the nonproducti ve
binding of pyruvate to LDH (20) and the condensationsof pyruvate with itself and
NA D ( 10) . B ot h M g t* a n d L D H c a ta l y z eth e enol i zati onof pyruvate and thus the
n u c leophilic add i ti o n o f e n o l p y ru v a te to C --1of N A D (21). B ecausegl yoxyl ate
c a n n o te n o l i z e .i t s h o u l dn o t u n d e r g oc o n d e n s a t i o nass p y r u v a t ed o e s .A l s o . s i n c e
g l y o x y l a t ee x i s t s i n a q u e o u ss o l u t i o na s t h c h y d r a t e .i t s h o u l d b e l e s s s o l u b l ei n
o r g a n i c s o l v e n t st h a n p 1 ' r u v a t ea n d t h u s m o r e e a s i l y ' s e p a r a t efdr o m p r o d u c t s .
A p o t e n t i a lp r o b l e m w i t h t h e u s e o f g l y o . r y l a t e h. o u ' e v e r ,w a s t h e N A D - l i n k e d
o x i d a t i o n o f g l y o x y l a t e b y L D H ( 1 4 , 2 2 ) . A t h i g h p H ( p H 9 ) , L D H o x i d i z e st h e
h ydr at e of gly o x y l a te a s a n a n a l o g o f l a c tate, E q . l 2l . Thi s reacti on nor onl y
co ns um esNA D b u t a l s o g e n e ra te so x a l a te ,whi ch i s a noncompeti ti vei nhi bi tor of
L D H wit h r es pe c tto p y ru v a te (a n d p re s u mabl ythe oxo form of gl y,orl ' l atet(2J).
NAD
g
lrAcoo-
-r4.*
?'
*o^coo-
NADH
!_-/
LDH
o
L2l
,-,oAaoo-
R E S U L T SA N D D I S C U S S I O N
Our work began with a search for substratesof LDH other than pyruvate which
would be good oxidizing agents for NAD regeneration.We measured the kinetic
r C o s t a n d s p e c i f i ca c t i v i t y o f L D H v s G I u D H a r e
$ 0 . 7 1 l 1 0 0 0U a n d 1 0 0 0U / m g v s $ 4 . 7 / 1 0 0 0U a n d
4 0 U / m g . r e s p e c t i v e l y . F M N r e d u c t a s e( 5 0 U / m g ) r e q u i r e s i s o l a t i o n ( R e f . ( 9 ) ) . P r i c e s ( S i g m a . 1 9 8 8 ) a r e
f o r e n z y m e s a s r e s e a r c hb i o c h e m i c a l sa n d t h e r e f o r e r e p r e s e n tu p p e r l i m i t s . O n e t h o u s a n d u n i t s ( l U :
I p m o l / m i n ) i s a p p r o x i m a t e l y t h e a m o u n t o f e n z y m a t i c a c t i v i t y r e q u i r e d t o g e n e r a t e1 m o l o f p r o d u c t /
day.
3 Prices of pyruvic (957o),glyoxylic (50% wlw
aqueous solution), and a-ketoglutaric g5%) acids are
7.6, 3.5, and 15 $/mol, respectively (Aldrich and ICN Biochemicals, 1988).
a R e d u c t i o n p o t e n t i a l sa r e r e l a t i v e t o
the half-reaction2H* + 2 e --->
H". for which E^: _0.42Y at
pH7.
402
CHENAULT AND WHITESIDES
.g
E
E
200
100
0
06121824
V / [ g l y o x y l a t e(] m L m i n - 1 )
F t c . L E a d i e - H o f s t e e p l o t u s e d t o d e t e r m i n e K ^ ( a p p ) o f L D H f o r g l y o x y l a t e i n t h e a b s e n c e( O ) a n d
presence (C) of I mn sodium vanadate.
constants of LDH with glyoxylate (pH 7.0, 25'C) and found the apparent
Michaelis constant,5K,,(app), to be 25 mr'a(Fig. 1). This value was in reasonable
agr eem entw i th p re v i o u s d e te rm i n a ti o n s(13-15) and w as si gni fi cantl yhi gher than
t h a t f o r p y r u v a t e( K , , : 0 . 1 5 - 0 . 3 3 m M ) ( 1 3 , 1 5 , 2 5 ) .T h e m a x i m a lv e l o c i t y( y , u * )
f or t he r edu c ti o n o f g l y o x y l a te w a s a p p r oxi matel ythe same as that w i th pyruvate
and was at l e a s t l 0 a ti me s fa s te r th a n th e oxi dati on of gl y' oxi rl atc(ne detectedno
oxidation with 50 mv glyoxylate and 2.8 mr'aNAD). At concentrations above -50
mu, glyoxylate inhibited its own reduction by LDH.
Since glyoxylate exists in aqueous solution as the hydrate and since vanadate
has been shown to catalyze the hydration/dehydration of glyoxylate (25), we also
measured the kinetic constants of LDH with glyoxylate in the presenceof I mrra
v anadat e( F i g . l ). T h e K,,,(a p p )w a s re duced to 8 mv. but V n,,,,al so dropped by
near ly half. In th e p re s e n c eo f l 0 mH rv anadate.LD H l ost al l acti vi ty. W e di d not
pur s ue t he m e c h a n i s m o f th i s i n h i b i ti on nor the further use of vanadate as a
c at aly s t in N AD -re g e n e ra ti n gs y s te ms .
Monomethyl oxalate (1) and S-methyl thiooxalate (2) were tested as pyruvate
analogs ,bu t n e i th e r w a s a s u b s tra teo f LD H (V < 0.005%of that for pyruvate).
o
o
tl
cH.s^coo2
tl
^\/n3\-./
, , ^ A ^ ^UUU
^1
ScsEuE I
To measure the stability of the pyruvate (glyoxylate)/LDH system, we incubated 0.1 mrraNAD and 50 mM pyruvate (glyoxylate) with and without LDH. The
activities of the speciesin solution were determined periodically by the enzymatic
assay. In all tests, the organic acids and enzyme remained fully active after 120h.
5 The K- measured is an apparent value because it is based on the total concentration of glyoxylate,
regardless of the form(s) it exists in and the species bound by the enzyme. The actual K- is probably
much smaller since glyoxylate exists in solution almost completely as the hydrate and yet LDH binds
the free aldehydic species as the substrate for reduction.
LACTATE DEHYDROGENASE-CAT ALY ZED REGENERATI ON
403
In the presenceof pyruvate, active NAD decreasedsomewhat: After 120h, in the
presence and absence of LDH, 45 and 60Vo,respectively, of the original active
NAD remained. With glyoxylate, in the presence and absence of LDH, 82 and
8970,respectively, of the original active NAD remained after 120 h.
LDH, coupled with pyruvic and glyoxylic acids as oxidants, successfullyregenerated NAD in oxidations of 4 mmol of glucose 6-phosphate (G6P) to 6-phosphogluconatecatalyzedby glucose-6-phosphatedehydrogenase(G6PDH) Eq. t3l.
The G6P/G6PDH system was chosen for initial demonstrationsbecausethe oxidation of G6P is thermodynamically favorable (spontaneoushydrolysis of 6-phosphogluconolactonerenders it irreversible) and not subject to product inhibition by
w as i sol ated as i ts bari um sal t (26) i n
6- phos phogluc o n a te6. -Ph o s p h o g l u c o n a te
(TTN)6
(J) for NAD(H) were 930-1070. At
70-80% yield. Total turnover numbers
the end of the reactions (4-6 h), both enzymes remained fully active, and the
nicotinamide cofactor retainedl6-99% of its original activity. In separatestability
studies, soluble G6PDH incubated in the presenceof 50 mM pyruvic or glyoxylic
acid (25'C, ambient atmosphere) remained 86 and707o active, respectively, after
95 h. When 50 mr',rG6P was included in the incubation mixture, G6PDH lost no
activity after 95 h.
aoPot2to,*\
.-:
NAD 1
bu'oH
,/
Qr-
n^coo-
\/
tou
/ I\
lrRDH/ \
3
n
n
\
l3l
^nnvvv
*,o
\*,"Nft:"
To compare the present method of NAD regenerationto methods using aKG/
GI uDH and F M N /O r. w e u s e d p y ru v a te /LD H to regenerate N A D for the
(3) to
HLA DH- c at aly z e d o x i d a ti o n o f c ' i s -1 ,2 -b i s(hydroxymethyl )cycl ohexane
(
4
,
(
T
a
b
l
e
( + ) - ( l R , 6 5 ) - c i s - 8 - o x a b i c y c l o [ 4 . 3 . 0 ] n o n a n - 7 -(o4n) ,eE q . [ 4 ]
l)
27). Both
d (29) LDH regeneratedNAD effiPAN-immobilized (28) and membrane-enclose
ciently and economically. Particularly striking was the stability of LDH, which
ffoH
\-A=,oH
3
I
n
v
tto
2 NAD
\/
2 NADH-/\
,-
I ton
\
2 n^cooO
2 ,A.oo-
\-ry
4
6 TTN : mol of isolated product/mol of catalytic species (enzyme or cofactor) in reaction.
t4l
404
C H E N A U L TA N D W H I T E S I D E S
TABI-E I
C o m p a r i s c o' rfnM e t h o d so f R e g e n c r a t i nNgA D t l o m N A D H l b r t h c O r i t l l r t i o no l ' 3 t o 4
Catalyzeb
c ly H I . A D H
Pvruvate/LDH
Regenerating system
Oxidant (mmol)
Enzyme (units)
Millimolesof NAD
Synthetic system
Millimolesof 3
Units of HLADH
R e a c t i o nt i m e ( d a y s )
Product yield (Vc)'
Pyruvic acid
(l7D
LDH (410)
0.12
TTN.
NAD
R e g e n e r a t i n ge n z y m e
HLADH
Recovered activit "v (% \
NAD
R e g e n e r a t i n ge n z - v m e
HLADH
$imol NAD regenerated'1
71
-520
1.5
85
1000
3.9 x 106
1.7x 104
11
'
l-s0
II0"
14
Glvoxvlate/LDH
aKG/GluDH"
FMN/O.n
Glyoxylic acid
( 170 add.ed
.or
contrnuously)''
a-Ketoglutaric
a c i d ( l - 5 3)
FMN (20.3)
GIUDH (230)
0.27
1.2
69
2-50
1.5
U-i
14
80
l.s
liO
430
l0
1.9x 104
60
l0
]]
. rI
l.l x l0l
150
" Data taken from Ref. (4).
h Data taken from Ref. (26).
' Based on isolated.distilled product.
d B a s e d o n t h e t o t a l c o s t o f N A D . o x i d a n t . a n d r e g e n e r a t i n ge n z y m e ( o n e - t i m e u s e ) .
' G l y t l r y l a t e c l e a c t i v l t c dH L A D H .
t G r i n d i n g o f t h e i m m o b i l i z a t i o n g e l i n t o s m a l l e r p a r t i c l e sb y t h e s t i r b a r i n c r e a s e ctlh e s L t d i r c ca r e a o f
t h e g e l a n d t h u s t h e a p p a r e n ta c t i v i n ' o f t h e i m m o b i l i z e dL D H .
. This irctivlrtioo
e l c ( t h l e c t l ' i i r l s )I.t s
n l ' F l l - , A I ) H( p u r eh l r s c t il r s l r o n h i l i z c t lp o r i t l c r )r i i r \ r . c t l ( ) ( l ui h
origin is unknou'n.
was greater than that of GIuDH (4). That LDH and GIuDH are cytoplasmic and
m it oc hondri a le n z y m e s , re s p e c ti v e l y .may accountfor thi s di fferencei n stabi l i ty.
M it oc hondri a l e n z y m e s te n d to re q u i re associ ati on w i th mi tochondri al membr anesf or sta b i l i ty a n d a re l e s s s ta b l e i n sol uti on than cytopl asmi c enzymes.
W hen gly o x y l a te (i n i ti a l c o n c e n tra ti o n: 0.23-0.25 u) servedas the oxi dant i n
Reaction [4], however, no product formed. Recovered enzymes showed nearly
f ull LDH a c ti v i ty b u t n o H L AD H a c ti vi ty. l n subsequentstabi l i ty studi es, gl yoxylate irreversibly deactivated HLADH by a process that was neither first- nor
second-orderTwith respect to the enzyme (Fig. 2). Glyoxylate probably caused its
deactivation by condensingwith the arginine in the binding domain of HLADH, in
analogy to the deactivation of HLADH by 2,3-butadioneand phenylglyoxal (30).
7 HLADH
exists as a dimer.
LACTATE DEH Y DROG EN AS E-C AT ALY ZED REG E N ERATIO N
405
I U\J
BO
.>
60
C)
(J40
o\
20
0
40
t,h
F t c . 2 . E f f e c t s o f 0 ( C ) , 0 . 5 ( A ) , 5 . 0 ( f ) , a n d 5 0 m t ' r ( O ) g l y o x y l a t e a n d - 5 0m v g l y o x y l a t e p l u s 5 0 m r r . l
cyclohexanol (I) on HLADH.
Cyclohexanol (50 mu) slightly improved the stability of HLADH in the presence
of 50 mM glyoxylate. A final attempt at using glyoxylate in Reaction [4] in which
glyoxylate was added to the reactor (2.5 mmol h-r) also failed. After 4 h, HLADH
retained only 40Voof its activity.
The advantagesand disadvantagesof using pyruvate and glyoxylate with LDH
to regenerate NAD are summarized in Table 2. Pyruvate/LDH is perhaps the
simplest, least expensive, and most stable system for regeneratingNAD for reactions in which the oxidation step is thermodynamically favorable (or can be made
so by r em ov ing o r re a c ti n gfu rth e r th e p ro d uct). W i th enzvmes that are not susce pt ible t o deac ti v a ti o n b y g l y o x y l a te . g l y o xyl ate offers advantagesof a hi gh
r e d u c t i o np o t e n t i a la n d a n e v e n l o w ' e r - c o st th a n p v r u v i r t e .A d i s i r c l v ' u n t a og le' l - l ) H
i s i t s i n a b i l i t yt o o x i d i z eN A D P H . a n d c r K G / G l u D H r e n - r a i nt sh e o n l y e n z y m a t i c
m e t h o d c a p a b l e o f d i r e c t r e g e n e r a t i o no f N A D P . a K G h a s a g o o d r e d u c t i o n
p ot ent ial. I n s om e re a c ti o n s ,th e z w i tte ri o n i c product, r,-gl utamate,may be easi er
to s epar at ef r om p ro d u c ts th a n l a c ti c o r g l y col i c aci ds. Methods based on LD H
a n d G I uDH ar e s u p e ri o r to th o s e b a s e d o n the reducti on of FMN .
T AB L E2
Advantages
andDisadvantages
of UsingPy.rffiTX$,YoxylicAcidsr.,,,ith
LDH to Regenerate
Method
Pyruvate/LDH
Glvoxvlate/LDH
Advantages
Inexpensive oxidant
High specific activity of enzyme
Inexpensive enzyme
Small K^ for pyruvate
Compatibility with other enzymes
Very inexpensiveoxidant
High specific activity of enzyme
Inexpensive enzyme
Strong oxidizing potential
Compatibility with NAD
Disadvantages
Moderate oxidizing potential
M i l d d e a c t i v a t i o no f N A D b y p y r u v a t e
Inability to oxidize NADPH
Large K^ for glyoxylate
Deactivation of HLADH
(and other enzymes'l)
Inabilitv to oxidize NADPH
406
CHENAULT AND WHITESIDES
EXPERIME,NTAL
Materialsand methods.LDH (rabbitmuscle,ammoniumsulfatesuspension),
HLADH
ammoniumsulfatesuspension),
G6PDH (Leuconostocmesenteroide.r,
(lyophilizedpowder), and biochemicalsfrom Sigmawere used without further
(98%),pyruvic acid (98%),
purification.cis-1,2-Bis(hydroxymethyl)cyclohexane
and glyoxylic acid (50%w/w aqueoussolution)from Aldrich were usedwithout
further purification.Water was doubly distilled,the secondtime througha Corning AG-1bglassstill. The autotitratorassemblyconsistedof a RadiometerPHM82
pH meter, TTT80 titrator, TTA80 titration assembly,ABU80 autoburette,
REA160 titrograph,and REA270 pH stat unit. rH and rrc NMR spectrawere
usingCHCl.r('H 6 7.26, t3C
takenon Bruker AM-300and AM-250spectrometers,
(methyl rH 6
6 77.0 in CDCI3)or sodium3-(trimethylsilyt)-l-propanesulfonate
0.00, r3C6 0.00 in DzO)as internalreferences.Infraredspectrawere taken on a
Elementalanalyseswere performedby
Perkin-Elmer 598 spectrophotometer.
SpangMicroanalyticalLaboratory.
observaEnzymaticassays.All assayswere basedon the spectrophotometric
:
(e
mu-r
6.18
nm
NADH
at
334
of
tion of the appearanceor disappearance
containing
a
solution
cm-r). For LDH, the assaywasinitiatedby adding0.01ml of
e E A ) b u f f e r .p H 7 . 6 .
0 . 0 1 - 0 . 1U o f L D H t o 3 . 0 0m l o f 0 . 1 v t r i e t h a n o l a m i n( T
l A D H . A s s a y ' o fL D H w i t h
c o n t a i n i n g 2 . 3m u s o d i u mp y r u v a t ea n d 0 . 2 3m r n N
exceptin 0.I rt Hepcstlr Mops/
glyoxylateas substratewas performedsimilarl-'N a O H b u f f e r ,p H 7 . 0 ,w i t h 5 0 m l r g l y o x y l a t eF. o r H L A D H . 0 . 0 1 m l c o n t a i n i n g
0 . 0 1 - 0 . 1U o f H L A D H w a s a d d e dt o l . l 0 m l o f 0 . 1 r ' aG l y - G l y b u f f e r .p H 8 . 0 .
containingg.gmu cyclohexanoland 0.50 mr'aNAD. Units were expressedas
activity with 3, which has a maximalvelocitythat is 80%of that of cyclohexanol
(27).For G6PDH,0.02ml containing
0.01-0.10U of G6PDHwasaddedto 2.98ml
9.8 mv G6P,6.9 mna
buffer,pH 7.0or 7.6, containing
0.1 rvrpotassiumphosphate
up to
by adding0.10ml containing
NAD, and 3 mrurMgCl2.Pyruvatewasassayed
0.33mlt NADH
210nmolof pyruvateto 0.96ml of 0.I r'aTEA. pH 1.6.containing
and2 U of LDH. Glyoxylatewas assayedby'the methodof Bergmeyerand Lang
andNADPH, respec(J1),substituting
LDH andNADH for glyoxylatereductase
tively. Total NAD(H) was assayedby the methodof Bernofskyand Swan(32),
and oxalatewas assayedusinga diagnostickit from Sigma.
Monomethyloxalate. Reactionof oxalic acid (10 g) and methanolin carbon
tetrachloride(JJ) gave,after distillation,267odimethyloxalateIsubl.40'C (0.1
1n d2 7 %
T o r r ) ; ' H N M R ( C D C l r )6 3 . 8 9( s ) ; r 3 CN M R ( C D C | - .6) , 5 3 . - 5l -.s 7 . 8 a
(0.2Torr)1,'H
monomethyloxalate:bp 50-55'C(0.1Torr) Uit. (JJ) bp -50--s2"C
N M R ( C D C h )5 3 . 9 4( s , 3 H ) , 1 0 . 5 7( s , I H ) ; r 3 CN M R ( C D C l r )6 - 5 4 . 21, 5 8 . 2 ,
195.5.
PotassiumS-methylthiooxalate.Oxalyl chloride(10 mmol) was convertedin
(30%):rH NMR (DrO)E 2.33(s).
S-methylthiooxalate
four steps(34)to potassium
Enzymestabilities.Pyruvic or glyoxylicacid (50 mu) and NAD (0.1mu) were
incubatedwith and without LDH (l U) in 0.10 u TEA buffer, pH 7.6,25'C.
Periodicaliquotswere assayedfor activitiesof the organicacid, NAD, and LDH.
G6PDH(2U) was incubatedin 50 mv Tris/HClbuffer,pH 7.5,6.3mu MgCl2,
LACTATE DEHYDROGENASE-C
AT ALY ZED REGENERATION
407
25"C, in the presence of 0, 5, 15, and 50 mM pyruvate or glyoxylic acid. G6PDH
was similarly incubated with 50 mM organic acid plus 50 mr'a G6P. Periodically,
aliquots were assayed for enzymatic activity.
HLADH (0.5 mg) was incubated in 0.1 rraMops/NaOH buffer, pH 7.0, 25"C in
the presence of 0.0, 0.5, 5.0, or 50 mrr,lglyoxylate, with and without 50 mv
cyclohexanol. HLADH was similarly incubated with 50 mM pyruvate, with and
without cyclohexanol. Periodically, aliquots were assayedfor enzymatic activity.
Oxidation of G6P using LDH to regenerate ltlAD. Each reaction contained in23
ml of water: 4 mmol of G6P (disodium salt), 4 mmol of pyruvate or glyoxylate, 3
pr.molof NAD,75 p.mol of DTT, and 75 pmol of MgCl2. Soluble LDH (125 U) and
G6PDH (200 U) were added to initiate the reaction. Reaction progress was monitored by recording the volume of 1.000 N NaOH delivered by an autotitrator
assembly to maintain constant pH (pH7.6 with pyruvate, pH 7.0 with glyoxylate).
Upon completion of the reaction, the mixture was assayedfor G6PDH, LDH, and
NAD and oxalate if glyoxylate had been used as the oxidant. BaClr ' 2H2O (6
mmol) and then 8 ml of ethanol were added to the stirred solution. The precipitate
was filtered, dried in uac'uo, and assayed for 6-phosphogluconateas described
p r ev ious ly ( 25) .
(3) using LDH to regenerOxidation of cis-l ,2-bis(hydroxymethyl)c-vc'lohexane
ate llAD. A 3-liter, three-neckedround-bottomed flask with magnetic stirring bar
w as c har gedwith 3 ( 1 0 .5g , 7 | mmo l ), p y ru v i c aci d ( 12.2 ml , 172mmol ) di ssol ved
i n 150 m l of wate r p l u s 4 5 ml o f 4 N N a O H , 3 7.5 ml of 0.1 r' aGl y-Gl y buffer. pH
8.0, 520 ml of nitrogen-sparged water, HLADH (290 mg. -520U). and PANi mm obiliz ed ( 27 ) L D H (4 1 0 U , 1 0 0 ml o f g el ). A l i ttl e 4 N N aOH w as added to
a d jus t t he pH t o 8 .1 , a n d th e m i x tu re w a s spargedw i th ni trogen for 30 mi n to
re m ov e diox y ge n . A 5 0 -p l a l i q u o t w a s a s s a y edfor i ni ti al pyruvate concentrati on.
NA D 0. 12 m m o l ) w a s a d d e d to th e m i x tu re . and 50 pclw as removed to assayfor
i n it ial NA D( H) c o n c e n tra ti o n . H e x a n e (l l i ter) w as l ayered over the aqueous
solution. and the reaction was stirred gently. The progress of the reaction was
monitored by assayingfor pyruvate. After 40 h, the reaction was complete. Enzym at ic as s ay s ho w e d 1 1 0a n d 7 7 % o f th e o ri g inal H LA D H and N A D (H ) acti vi ti es,
re s pec t iv ely .r em a i n i n g . T h e h e x a n e w a s re m oved by steel cannul a and concentrated to a yellow oil. The gel was allowed to settle and most of the aqueouslayer
removed by cannula. The gel was centrifuged and the supernatantdecanted. The
gel was then washed twice by resuspensionin water, centrifugation, and decanting. All aqueous layers were combined and extracted with ether. Ethereal portions wer e c om b i n e d w i th th e c o n c e n tra te dh exane sol uti on, dri ed over MgS Oa,
and evaporated under reduced pressure. Distillation of the crude product gave 8.5
(4) as a col orl essoi l :
g ( 85%) of ( + ) - 1 IR , 6 5 )-c i s -8 -o x a b i c y c l o [4 . 3.0]nonan-7-one
b p 6 5 - 7 5 ' C ( 0 . 3 T o r r ) ; a 2 ; + 4 3 . 8 ' ( n e a t ) ; ' H N M R ( C D C I 3 )6 l . l - 2 . 6 ( m , 1 0 H ) ,
: 8.8 H z, I H each);i r
3 . 90and 4. 15 ( 2 d d , " Iu i .: 1 .2 a n d 5 .0 H 2 , re s p ecti vel y,.,Ig.,n
(neat ) 1770 c m r. An a l y ti c a l d a ta w e re i n a g reementw i th the l i terature (4,27).
The LDH gel assayed for 150% of its original activity.
A similar reaction was run using HLADH and LDH enclosed in dialysis membrane tubing (29).Yield of distilled product was 88%.
Attempted oxidations of 3 using glyoxylate and PAN-immobilized or mem-
408
CHENAULT AND WHITESIDES
brane-enclosed
LDH were identicalto the reactionsdescribedaboveexceptthat
glyoxylatereplacedpyruvate. In a final attempt,a neutral solutionof sodium
glyoxylate (l .14u) was addedat a rateof 2.2 ml h- I to a biphasicsystemcontaining 10g of 3, 20 mrrltriethanolamine,
and0.12mmol of NAD in 800ml of degassed
water. HLADH (500U) and LDH (1500U) were enclosedin dialysistubing(8 ml
total volume)and submergedin the aqueouslayer, which was then overlayered
with hexane.
ACKNOWLEDGMENTS
This work has beensupportedin part by NtH Grant GM 30367and by a grantfrom Firmenich.SA.
(Grant 5-T32H.K.C. gratefullyacknowledgessupportas an NIH TrainingGrant Trainee. 1984-8-5
GM-07598).
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