Principles of Bioinorganic Chemistry - 2003
Lecture
1
2
3
4
5
6
7
8
9
10
11
12
13
14
*Makeup
Date
Lecture Topic
Reading
n+
9/4 (Th)
Intro; Choice, Uptake, Assembly of M Ions
Ch. 5
9/ 9 (Tu)
Metalloregulation of Gene Expression
Ch. 6
9/11 (Th)
Metallochaperones; Metal Folding, X-linking
Ch. 7
9/16 (Tu)
Zinc Fingers; Metal Folding; Cisplatin
Ch. 8
9/18 (Th)
Cisplatin; Electron Transfer; Fundamentals
Ch. 9
9/23 (Tu)
ET Units; Long-Distance Electron Transfer
Ch. 9
9/25 (Th)
ET; Hydrolytic Enzymes, Zinc, Ni, Co
Ch. 10
10/ 7 (Tu)
Model Complexes for Metallohydrolases
Ch. 10
10/9 (Th)
Dioxygen Carriers: Hb, Mb, Hc, Hr
Ch. 11
10/10 (Fr)*
O2 Activation, Hydroxylation: MMO, P-450, R2 Ch. 11
10/14 (Tu)
Model Chemistry for O 2 Carriers/Activators
Ch. 12
10/16 (Th)
Complex Systems: cyt. oxidase; nitrogenase
Ch. 12
10/21 (Tu)
Metalloneurochemistry/MedicinalInorg. Chem.
10/23 (Th)
Term Examination
class, 8:30 – 10 AM; room to be announced
Problems
Ch. 1
Ch. 2
Ch. 3
Ch. 4
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 10
Ch. 11
Ch. 12
You should have your paper topic approved by Prof. Lippard this week, if you have
not done so already (by 10/12 please). The oral presentations will be held in
research conference style at MIT's Endicott House estate in Dedham, MA, on
Saturday, October 18. WEB SITE: web.mit.edu/5.062/www/
Hydrolytic Enzymes, Zinc and other Metal Ions
PRINCIPLES:
•M(OH)n+ centers supply OH- at pH 7 by lowering water pKa
•Mn+ serves as general Lewis acid, activating substrates
•Rate acceleration occurs by internal attack within coord. sphe
•Protein side chains greatly assist assembly of transition state
•Carboxylate shifts can occur, especially at dimetallic centers
•Electrostatic interactions predominate
•Non-redox active metal ions often but not universally used
Illustrating the Principles:
•Carboxypeptidase, carbonic anhydrase - delivering
hydroxide
•Alcohol dehydrogenase: an oxidoreductase
•Dimetallic metallohydrolases: are two metals better than
one?
Carboxypeptidase A: A Hydrolytic Zinc Enzyme
Reaction catalyzed:
R–CH–C(O)–NH–R’
R–CH–CO2- + +NH3–R’
NH2R’’
NH2R’’
Cleaves C-terminal peptide bonds; prefers aromatic residues.
Active site contains a single
catalytic zinc, essential for activity.
The glutamate can undergo a
carboxylate shift. Thermolysin has a
similar active site; it is an
endopeptidase.
Carboxypeptidase A
structure with the inhibitor
glycyl-L-tyrosine bound at
the active site. Note
hydrogen bonds to key
residues in the active site
that position the substrate
moiety for bond scission.
Catalytic Mechanism
for Carboxypeptidase
A
Summary of events:
1. Substrate binds; orients by the
terminal carboxylate.
2. Deprotonate bound H2O.
3. Polarize scissile bond by
Arg127.
4. Bound OH- attacks peptide
C(O).
5. Form tetrahedral transition
state.
6.Lose 2 peptide fragments and
recycle the enzyme.
Principles illustrated:
1. Zinc serves as template.
2.Metal supplies cleaving reagent,
OH-, and organizes key groups.
3. Chemistry achieved at neutral
pH! Kcat ~ 100 s-1 .
Carbonic Anhydrase, the First Known Zn Enzyme
Reaction catalyzed:
CO2 + H2O
H2CO3 ~ 106 s-1
Carbonic Anhydrase
PZn(OH2)2+
PZn(OH)+ + H+ Keq = 10-7 M = kf/kr
Note:
Rate 10-2 s-1 at pH 7; kf 106 s-1 in active site.
Paradox:
The reverse reaction is diffusion controlled, with kr ~ 1011 M-1 s
Thus kf ≤ 104 s-1. So how can the turnover be 106 s-1 ?
Answer:
Facilitated diffusion of protons by buffer components bound
to the enzyme.
Possible Carbonic Anhydrase Mechanism
Alcohol Dehydrogenase, an Oxidoreductase
Reaction catalyzed:
RCH2 OH + NAD+
RCHO + NADH + H+
Enzyme contains two 40 kDa polypeptides, each with 2
Zn2+centers in separate domains. One zinc is structural, the
other catalytic.
Catalytic zinc is 20 Å
from the surface, near
the nicotinamide
binding region. This
center is not required
for NAD + cofactor
binding. Alcohol
substate DO require
zinc and bind directly
to the metal center,
displacing the
coordinated water.
Schematic Diagram
NAD+ binding to the
active site of LADH,
with specific, wellpositioned amino
acid side chains
holding it in place.
Ethanol is shown
bound to the zinc,
displacing water. The
system is set to
undergo catalysis.
Hydride Transfer Mechanism
R'
+
H
N
R
Zn
H
H2 N
O
O
H2 O
R'
..
N
H
+ RCHO +
H
H2 N
O
Zn
H2 O
Note hydride transfers from a-C of
alcohol to nicotinamide ring.
Dinuclear Metalloenzymes
Redox-active dinuclear
Metalloenzymes:
Methane monooxygenase (Fe 2)
Tyrosinase (Cu 2)
Catalase (Mn 2)
Isomerase:
Peptide hydrolases:
Xylose isomerase (Mg 2)
Phosphoester hydrolases:
Ser/Thr phosphatases (Fe/Zn or Fe/Fe)
Alkaline phosphatase (Zn 2)
Nuclease P1 (Zn 2)
Inositol Monophosphatase (Mg 2)
RNase (Mn 2 and Mg 2)
DNA polymerase I (Mg 2)
Other metallohydrolases:
Arginase (Mn 2, Co 2)
Urease (Ni 2)
-Lactamase (Zn 2)
Methionine aminopeptidase ( Zn 2 or Co 2)
Leucine aminopeptidase (Zn 2)
Alkaline Phosphatase; a Dizinc(II) Center Activates the Substrat
1.
3.
2.
1. The substrate binds to
the dizinc center; a
nearby Arg also helps
activate it.
2. A serine hydroxyl group
attack the phosphoryl
group, cleaving the ester.
The phosphate is
transferred to the enzyme,
forming a phosphorylserine residue.
3. Hydrolysis of this
phosphate ester by a zincbound hydroxide completes the catalytic cycle.
This mechanism is
supported by studies with
chiral phosphate esters
(ROP18O17O16O)2-; there is
no net change in chirality
at phoshorus.
pKa Values of Metal-Bound Water for Common
Metal Ions in Aqueous Solution
M OH2
pKa
M OH + H+
Fe3+ -OH
Cu2+ -OH
pK a
Dimetallics can move the
value into the physiological
range near pH 7
Zn2+ -OH
Co2+ -OH
Ni2+ -OH
Mn2+ -OH
Mg2+ -OH
1
2
3
4
5
6
7
8
9
10
11
Barnum, D. W. Inorg. Chem. 1983, 22, 2297.
12
13
14 pH
Advantages of Carboxylate-Bridged Dimetallic Centers
in Chemistry and Biology
Modes of Substrate (S) Attack by an Activated Nucleophile (N) at a
Carboxylate-Bridged Dimetallic Center
O
O
C
R
MB
MA
B
O
C
R
O
N
S
MA
MB
O
C
O
C
R
:
MB
MA
A
S
:
:
S
N
:
:N
N
MB
MA
O
D
C
R
S
N
S
MB
MA
O
O
E
O
C
R
Carboxylate Ligation in Metalloproteins
and the Carboxylate Shift
Biologically available carboxylates:
O
-
O
O
-
O
O
C
C
H 2C
OH
Carbonate
-
H3N
C
COO
H2 C
-
H
Aspartate (Asp) D
Carbonate is encountered in transferrin
Lys* is found in urease and rubisco
H3 N
C
O
-
O
O
C
C
CH2
NH
H2 C
CH2
COO-
H
H2 C
Glutamate (Glu) E
H3 N
CH2
C
COO-
H
Lys* Carbamate
Chemistry Leading to the Definition of the Carboxylate Shift
methanol
M(O2CCH 3)2 + BIPhMe
M = Mn, Fe
R
R
C
C O
O R O
C
N
O
O
O
N
M
M
M
N
O
O
O
N
C
O R O
O C
C
R
R
[M3II (O2CCH 3)6(BIPhMe)2]
"This movement, which we term 'the carboxylate shift,'
may be of general importance in the active site structures
of metalloproteins."
Rardin, Bino, Poganiuch, Tolman, Liu, Lippard
Angew. Chem. Int. Ed. Engl., 102, 812 (1990)
R
O
O
M
R
M
O
O
M
M
R
O
M
M
O
The Expanded Carboxylate Shift
C
O
M
O
M
M
O
•
•
C
O
M
C
O
O
C
O
R
M
M
M
M
M
M
•
•
M
R
O
C
O
O
M
R
O
M
O
R
C
M
M
O
C
O
•
•
R
C
O
M
O
M
R
M
C
O
•
•
R
R
R
O
•
•
M
C
O
O
•
•
R
M
Note syn/anti lone
pair traversals
The Active Sites of Selected Dinuclear Metalloenzymes
Catalyzing Hydrolysis of Biological Substrates
Asn150
Asp45
H2N
H2O
Fe
N
HN
O
Asp90
O His281
H
O
H2O
His92
O
Zn
O
Trp1
O
N
N
O Asp118
NH2
NH
O Zn
N
O
His6
HN
His199
N
H
O
H
O
His60
N
Zn
NH
N
O
N
H
His116
Asp122
Nuclease P1
Calcineurin
Asp301
His97
O
His201
HN
N
N
His230
HN
O His57
H
O
Zn
Zn
O
N
NH
N
N
O
Glu152
HN
O
N
H
Asp179
His55
O
O
H
O
Zn
O
Zn
O
HN
Lys169
Phosphotriesterase
O
N
N
H
Asp117
Aminopeptidase
His156
Structure and Chemistry of Arginase
NH 2+
+
H3 N
CO2-
N
H
NH 2
+H N
3
+ H2 O
CO2L-Ornithine
L-Arginine
O
+
H2 N
Urea
Asp-232
His
O
Asp
O
HN
N
O
Mn
+
O
Asp-128
NH 3+
O
2
H
O
Asp-124
O
Mn
Active Site of Arginase
O
2+
N
O
His
NH
NH 2
Postulated Catalytic Mechanism for Arginase
O
NH 2
N
H
Mn2+
I
O
O
O
NH 2
O
NH 2
O
O
H Mn2+
O
O
O
N
NH 2
H
O
Mn2+
Mn2+
H
O
II
Asp 128
Asp 124
NH 2
+
H
L-Ornithine
H2O
L-Arginine
Mn2+
H2
O
O
NH 2
Mn2+
Mn2+
O
O
NH 2
Mn2+
Christianson, 1996
III
IV
Urea
Principles illustrated: the dimetallic affords hydroxide; the
substrate is positioned by residues in the active site; the dimetallic
stabilizes the urea leaving group; redox inactive metal;
The Dinickel(II) Metalloenzyme Urease
History of Urease
1926, Sumner crystallizes urease
O
1975, Blakeley and Zerner discover
that urease is a dinickel enzyme
N
N
O
N Ni
1995, Hausinger and Karplus determine
X-ray structure; unusual active site
N
N
O
Ni
O
N
Urea Hydrolysis
O
H2N
NH2
H2 O
urease
O
H2N
OH
+ NH3
NH3 + H2 O
N
O
N
N
Native and Inhibited Urease from B. Pasteurii
Lys220*
His275
HN
HN
HN
O
N
Ni 1
N
O
H
N
His137
N
Ni 2
O
H(2)
OH2
H2O
His249
N
H
O
O
His139
N
Asp363
Native urease, 2.0 Å resolution
His
HN
HN
275
Lys220*
HN
H
N
His137
N
O
O
N
Ni1
Ni2 N
N
S
N
HO
O
H
249
3
His
O Asp 63
His
HN
His139
-Mercaptoethanol inhibited urease,
1.65 Å resolution
HN
Lys220*
275
HN
N
O
Ni1
O
N
H
N
His137
Ni2 N
O
N
O P NH O
N
2
H
His249 NH
363
Asp
2 O
His139
DAP inhibited urease, 2.0 Å resolution
Benini et. al.Structure 1999, 7, 205-216.
Benini et. al. JBIC 1998, 3, 268-273.
Proposed Mechanism of Urea Hydrolysis
O
CO2 + (2)NH3
Ni
H2O
H2N
Ni
NH2
OH
Ni
Ni
O
Ni
O
Ni
OH
NH2
O
H2N
NH2
O
O
NH3
Other urease substrates:
O
H 2 N NHCH3
O
H 2 N NHOH HOHN NHOH H NH 2
O
NH 2
Alternative Mechanism of Urea Hydrolysis
O
CO2 + (2)NH3
Ni
H2 O
H2 N
Ni
NH2
OH
Ni
Ni
Ni
O
OH2
Ni
OH
C
O
H2 N
N
NH3
NH2
Metallo--lactamases, an Emerging Clinical Problem
PZn(OH2)2+
PZn(OH)+ + H+
R'
Reaction Catalyzed:
R'
S
C
H2 O
N
R"
O
S
C
HN
OH
H86
H2O
Zn2
Zn1
OH
O
O
D90
Bacillus cereus
Zn...Zn, 3.5 and 4.4 Å
H210
H84
Zn1
H160
R"
COOH
C168
H149
H88
O
COOH
Active Sites:
H86
Keq = 10-7M = kf/kr
H
O
H162
D88
Zn2
H2O
H225
H89
H101
H
O
Zn1
H99
C181
Zn2
OH2
H223
D103
S185
Stenotrophomonaso maltophilia
Zn ...Zn, 3.4 Å
Bacteroides fragilis
Zn... Zn, 3.5 Å
-Lactamase from Bacteroides fragilis
H99
D103
Wat1
H223
Zn2
Zn1
H101
Wat2
C181
H162
N.O. Concha, B.A. Rasmussen, K. Bush, O.
Herzberg (1996), Structure 4, 823-836
Active Site of a -Lactam Antibiotic
Resistance Enzyme, IMP-1 Metallo- -lactamase
(Fitzgerald, et al., 1999 )
His145
HN
Cys164
N
S
His206
HN
N
NH
Zn
Zn
O
H
O
O
Asp86
N
N
His84
His82
NH
Possible Mechanism for Metallo--lactamases
O
O
Zn
Zn
HO O
O
Zn
Zn
O
O
R1
S
OH
O
O-
N-
O
N
O
OH
NH
O
R1
R2
S
R2
S
blue intermediate
O
O
S
O
-
R2
R1
O
NH
6 S
5
4N
O
-9
COO
1
400 nm
NH
10
12
NO2
H2 O
S
S
O
OH HN
COO496 nm
NO2
nitrocefin: a substrate for investigating the mechanism
NO2
NO2
Summary - Points to Remember
•Both mono- and dimetallic centers lower the pKa value of
bound water, allowing hydroxide to be delivered at pH 7.
•Coordination of the leaving group portion of the substrate
to a metal ion activates the substrate for nucleophilic attack.
•Residues not coordinated but in the second coordination
sphere can participate directly (serine in phophatases) or
indirectly (arginine in alcohol dehydrogenase) in substrate
attack, orientation, and/or activation.
•Carboxylate shifts facilitate substrate binding, activation.
•Redox inactive metal ions (Zn2+, Ni2+, Mn 2+, Co2+) preferred.