BS 501

Molecular Enzymology

Narayan Punekar

nsp@
 Evaluation Scheme
Assignments & Problem Sets 20

Mid-Semester Examination 30

End-Semester Examination 50

Attendance 80 percent (XX Grade)

 Course Contours
Enzyme catalysis a perspective
Hallmarks of an enzyme catalyst
Enzyme kinetics the practice
Kinetic mechanisms
Chemical mechanisms
Frontiers in enzymology
Structural enzymology
 Reference Books
1] The Enzymes, PD Boyer (ed.) Vol. I and II, Academic Press, 1970
2] Enzyme Kinetics, IH Segel, Wiley 1970
3] Biochemical Calculations, IH Segel, Wiley 1970
4] Enzymatic Reaction Mechanisms, C Walsh, WH Freeman 1979
5] Enzyme Kinetics, KM Plowman, McGraw-Hill, 1972
6] Enzyme Structure and Mechanism, AR Fersht, WH Freeman 1985
7] Immobilized Enzymes, MD Trevan, Wiley 1980
8] An introduction to the study of Enzymes, H Gutfruend, Wiley 1965
9] Fundamentals of Enzyme kinetics, A Cornish-Bowden, Portland Press, 2004
10] Fundamentals of Enzymology, NC Price and L Stevens, OUP 1982
11] Biochemistry, D Voet and JG Voet, Wiley 1990 (Ch. 12-14)
12] Enzyme Kinetics: From diastase to multi-enzyme systems, CUP 1994
13] Enzyme Kinetics: A modern Approach, AG Marangoni, Wiley-Interscience 2003
14] Practical Enzymology, H Bisswanger, Wiley-VCH 2004
15] Comprehensive Enzyme Kinetics, V Leskovac, Kluwer/Plenum 2003
16] Introduction to Enzyme and Coenzyme Chemistry, TDH Bugg, Blackwell, 2004
17] Catalysis in Chemistry and Enzymology, WP Jencks, Dover, 1986
18] Enzymes- Biochemistry, Biotechnology & Clinical Chemistry, T Palmer
19] Enzymes- A Practical Intr. to Str., Mech. & Data Analysis, RA Copeland
 Background Expected
Chemical kinetics Fundamentals

Concepts of equilibrium and steady-state

Chemical reactivity and molecular interactions

Acid-base chemistry and catalysis

Enzymology is the domain where Chemistry significantly

intersects Biology
and Biology is at its quantitative best!
Author(s) Year Contribution
*
R. Reaumur 1751 Gastric digestion in birds
L. Spallanzani 1780 Digestion of meat by gastric juice Landmarks in enzyme studies
A. Payen & J. Persoz 1833 Amylase (diastase) activity (Enzymology classics)
J. Berzelius 1836 Catalysis as a concept
W. Kuhne 1867 Enzyme term defined
J. Takamine 1894 Patent on fungal diastase
E. Fischer 1894 Lock and key concept
G. Bertrand 1897 Co-ferment (coenzyme) conceived
P.E. Duclaux 1898 Enzyme names to end with suffix ase
V. Henri 1903 Hyperbolic rate equation
S.P.L. Sorensen 1909 pH scale and buffers
L. Michaelis & M. Menten 1913 Equilibrium treatment for ES complex
R.M. Willstatter 1922 Trager theory of enzyme action
G.E. Briggs & J.B.S. Haldane 1925 Steady state treatment for ES complex
J.B. Sumner 1926 Urease Purification and crystallization
H. Lineweaver & D. Burk 1934 Double reciprocal plot (1/v versus 1/S)

K. Stern 1935 First ES complex observed

M. Doudoroff 1947 Radioisotope use in enzyme mechanisms
A.G. Ogston 1948 Asymmetric interaction with substrate
L. Pauling 1948 Enzyme binds TS better than S
F. Westheimer 1951 Enzymatic hydride transfer (2H, 3H
used)
D.E. Koshland Jr. 1958 Induced fit hypothesis
Enzyme Commission 1961 Enzyme classification and nomenclature
D.C. Phillips et al 1962 First enzyme structure - Lysozyme
W.W. Cleland 1963 Systematization of enzyme kinetic study
J. Monod, J. Wyman & J.-P. 1965 Model for allosteric transitions
Changeux
R.B. Merrifield 1969 Chemical synthesis of ribonuclease A
S. Altman & T.R. Cech 1981 Catalysis by RNA molecules
 Year* Enzymology - Topic of study
Scientist
E. Fischer 1902-C Stereochemistry and lock and key concept
S. Arrhenius 1903-C Activation energy and catalysis
E. Buchner 1907-C Cell-free extracts and fermentation
A. Harden & H. von Euler 1929-C Coenzymes and fermentation
C. Eijkman & F.G. Hopkins
O. Warburg
1929-M
1931-M
Vitamins, nutrition and coenzymes
Respiratory enzymes
Nobel laureates who contributed
A. Szent-Gyorgyi 1937-M Fumarate catalysis of TCA cycle to the growth of enzymology
R. Kuhn 1938-C Vitamins and coenzymes
A. Fleming 1945-M Penicillin and Lysozyme
*Prize awarded that year for:
J.B. Sumner, J.H. Northrop & 1946-C Purification and crystallization of enzymes
M. Kunitz C Chemistry; M Physiology and Medicine
C. Cori & G. Cori 1947-M Enzymes of glycogen metabolism
H.A. Krebs & F. Lipmann 1953-M TCA cycle and Coenzyme A
L. Pauling 1954-C Secondary structure - helix; concept that
enzyme binds the transition state
H. Theorell 1955-M Oxidative enzyme mechanisms
A.R. Todd 1957-C Nucleotides and nucleotide coenzymes
F. Sanger 1958-C Insulin sequence through proteases
S. Ochoa & A. Kornberg 1959-M Nucleic acid biosynthesis enzymes
M.F. Perutz & J.H. Kendrew 1962-C Crystal structure of globular proteins
D. Crowfoot Hodgkin 1964-C Structure of vitamin B12
K. Bloch & F. Lynen 1964-M Cholesterol and fatty acid enzymes
F. Jacob, A. Lwoff & J. Monod 1965-M Genetic control of enzyme synthesis and
allostery
L.F. Leloir 1970-C Sugar nucleotides and carbohydrate biosynthesis
E.W. Sutherland Jr. 1971-M Enzyme and metabolic regulation by cAMP
C.B. Anfinsen, S. Moore & 1972-C Chemical structure - catalytic activity of
W.H. Stein ribonuclease A
J.W. Cornforth 1975-C Stereochemistry of enzyme reactions
W. Arber, D. Nathans & H.O. 1978-M Restriction endonucleases
Smith
F. Sanger 1980-C Nucleic acid sequencing with enzymes
R.B. Merrifield 1984-C Chemical synthesis of ribonuclease A
J.W. Black, G.B. Elion & G.H. 1988-M Inhibitors (enzyme) as drugs
Hitchings
S. Altman & T.R. Cech 1989-C Catalysis by RNA molecules
E.H. Fischer & E.G. Krebs 1992-M Protein kinases and protein phosphorylation
K.B. Mullis 1993-C Polymerase chain reaction
P.D. Boyer, J.E. Walker & J.C. 1997-C ATP synthase and Na,K-ATPase
Skou
A.H. Zewail 1999-C Detection/ existence of transition state
I. Rose (and others) 2004-C Ubiquitin-Protein degradation and isotope
exchanges in enzymology
Class 4
Exploiting Enzymes for Technologies

Available diversity used in food, pharma..

Immobilization for better/optimal use
Reagents, biosensors, synthesis
Targets for drug discovery
Function in organic solvents
Engineered enzymes directed evolution
De novo enzyme design
Heterologous expression large scale
Starch Industry
Cyclodextrins

Cyclomaltodextrin-D
-glucotransferase

D-Glucosidase Amylase

STARCH

Pullulanase
Amylase

Glucoamylase

Glucose isomerase
Glucose Fructose
 Digestion of FAT


Lipase
(involved in hydrolysis of triacylglycerols)


Inhibition
(lipase activity - enzyme assay)


Inhibitor Screen
(natural or synthetic chemical libraries)


Lipostatin
(active principle)
 O H

Pharma Industry N
S

O N
O H O

O
Penicilloic acid
O H Lactamase
N
S

N
O O

Penicillin G O

Penicillin acylase
O

OH
H2 N
S
+
Phenylacetic acid

N
O O

O
6-Aminopenicillanic acid

O H
N
R S
Semisynthetic
N O Penicillins
O
O O O
Cephalosporins
Immobilized Enzymes
E E E E
E E
E E
E E
E E
E E
E E
E E E E
E E
E E
E E
E E

E E E E E E

Entrapment Covalent binding

E E E E
E E
E E
E E
E E
E E
E E
E E E E
E E
E E
E E
E E

E E E E E E

Electrostatic attraction Cross-linking

 Hallmarks of an enzyme catalyst

A
Catalysis Allosteric
site

Specificity S

Regulation I
Enzyme Active
site

Cell is NOT a bag of enzymes

Mathews CK, J. Bacteriol.,175:6377-81
Class 5 Complex cellular metabolic network

Greater than 99% of these reactions are catalyzed

 Range of enzyme specificities

Enzyme discrimination (and Limits of biological

specificity!)
Cytochrome oxidase O2, CO, CN-
Nitrogenase N2, O2, H2O2, Acetylene
Superoxide dismutase O2-., Cl-, CN-
RuBP carboxylase CO2, O2
Valine tRNA synthetase Ile, Leu

Cofactor tuning
Redox potential Fe, Heme, FAD

Testing the Limits

CoAS-Ac, NAD(P)
 Some enzymes are more
discriminating than others, but it
seems fair to say that any
enzyme can be fooled if one goes
to enough trouble.

JM Reiner, 1959
 enzyme
meaning in yeast in Greek)
(

coenzyme, cofactor, prosthetic group

apoenzyme and holoenzyme

metallo-enzymes and metal-activated
enzymes
(KD range 10-8 M)
Avidin-Biotin, 10-15M (t1/2, 2.5 yrs)
From: Biochemistry by Voet & Voet
 Enzymes can be grouped based on
Occurrence and/or source

Nature of substrate on which they act

Based on cofactor requirement

Common functional context

Nature of the overall reaction catalyzed

The mechanism of reaction (Walsh Book; Gal epimerase)

IUPAC Enzyme Commission

Ligases, 151
Isomerases,
Oxido-
180
reductases,
Lyases, 462 1295

Hydrolases,
1476
Transferases,
1302
EC nomenclature: Representative classes

EC No. Systematic name Trivial name Reaction

1. OXIDOREDUCTASES: Loss or gain of electrons by substrates
1.1.1.1 Alcohol:NAD+ Alcohol Alcohol+NAD+ Aldehyde+NADH+H+
oxidoreductase dehydrogenase

2. TRANSFERASES: Transfer of a reactive group from a donor substrate to an acceptor substrate

2.1.2.1 L-Serine:tetrahydrofolate Serine hydroxy- L-Serine+tetrahydrofolate
5,10-hydroxymethyl- methyltransferase Glycine+5,10-Methylenetetrahydrofolate
transferase

3. HYDROLASES: Introduction of elements of water into a substrate

3.1.1.1 Carboxylic ester Carboxylesterase Carboxylic ester+H2O
hydrolase Alcohol+carboxylate

4. LYASES: Elimination of a group with double bond formation or addition of group to double bond
4.1.1.1 Pyruvate carboxy-lyase Pyruvate Pyruvate Acetaldehyde+CO2
decarboxylase

5. ISOMERASES: Intramolecular rearrangements

5.1.1.4 Proline racemase Proline racemase L-Proline D-Proline

6. LIGASES (SYNTHETASES): Joining of molecules by covalent bond formation

6.1.1.1 L-Tyrosine:tRNA ligase Tyrosyl-tRNA ATP+L-Tyrosine+tRNA
(AMP) synthetase AMP+pyrophosphate+L-Tyrosyl-Trna
 Origins of Enzyme Catalytic Power

TS
Kinetic Barrier: Catalyst

Thermodynamic Barrier: Catalyst
G
+ Energy
uncat

E nergy
G
cat

R e a c t io n c o o r d in a t e

G=H
G H-TS
S and GF=G
G GR
 Enzyme only hastens the approach to
equilibrium:
G unaffected

Glucose Fructose DHAP GA3P

G
(Free
energy)

Equilibrium
Equilibrium

Glucose Fructose DHAP GA3P

 The approach to equilibrium
Glucose Fructose Sucrose Glucose + Fructose
1 1

Glucose
Fructose
Glucose

Concentration
Concentration

Fructose

Sucrose

0 0
Time Time
 Series of binary collisions

Even with multiple reactant systems!

Only Zero or First Order

Complex reaction may be broken down into
many simpler ones

A X

k2>>k1 k!>>k2

A
G
X

P
k1 k2
A X P

Reaction coordinate
Intermediates may accumulate!
[A]0

P
A
k1 k2
A X P

0
Time
 Time scales in catalysis

Catalytic turnover
Enzyme binds ligands Covalent modifications

10-15 10-12 10-9 10-6 10-3 100 103 Seconds

Allosteric transitions Enzyme synthesis/degradation

Cellular metabolite transport

Viewing frame dead time

Theories of catalysis
 T S

G uncat

E nergy
G cat

R e a c tio n c o o r d in a te

Where:

x is Guncat = -RT ln Keq

y is Gcat = -RT ln Keq
z is GRxn = -RT ln Keq
________________________________________________________________________
Arrhenius Theory TS Theory

k = A e-Ea/RT k = [kT/h] e-G /RT
________________________________________________________________________
 k = A e-Ea/RT

k/A = e-Ea/RT

ln (k/A) = -Ea/RT

ln (A/k) = Ea/RT

Ea = (RT ln 10) log (A/k)

Example: at 25C, (RT ln 10) is 1.36 kcal/mole

And for A = 6x1012 sec-1

Activation energy of 3.8 kcal/mole corresponds to a k of 1010 sec-1

Activation energy of 10.6 kcal/mole corresponds to a k of 105 sec-1


-G /RT
k = [kT/h] e

k = [kT/h] e-H /RT e-S /R

Guncat = (RT ln 10) log [(kT/h)/k uncat]

Gcat = (RT ln 10) log [(kT/h)/k cat]

-Gcat /RT
k cat = [kT/h] e
G
k uncat = [kT/h] e- uncat /RT

(Guncat-Gcat)/RT
k cat/k uncat = 10

Example: If Guncat = 25.7 kcal/mole and

Gcat = 11.0 kcal/mole
Then,

k cat/k uncat = 10(25.7-11.0)/1.36

= 5 x 1010
 Origins of Enzyme Catalytic Power

Proximity and
Orientation Effects

Stabilization of
Transition State or Electrostatics
Reactive Intermediate

Enzyme Catalytic
G
)
Power (

General Acid-Base
Metal ion Recruitment
Catalysis

Covalent Catalysis
(Nucleophilic or
Electrophilic)
 1.Proximity and Orientation
(Circe Effect)
Carboxylate assisted hydrolysis of phenyl esters and corresponding rate constants

1 Phenyl acetate 2 Phenyl succinate 3 Phenyl maleate

O O O
C C C
H3C O O O
+ O
O
H3C O
C O O
O

k1 = 0.002 M-1 s-1 k2 = 0.4 s-1 k3 = 20.0 s-1

Ester hydrolysis by tertiary amine Ester hydrolysis by imidazole

NO 2 NO2
O O O O
C C C C
H3C O O O O
H3C
+ + N
N CH3 versus
H3C CH3 versus N
N CH3 NH
CH3 NH
 Conformational flexibility and enzyme catalysis

Conformational change Effect Example

Orbital steering and small Large changes in kinetic Chymotrypsin; NADP-isocitrate
structural changes property, co-operativity dehydrogenase; keto-steroid
isomerase; most enzymes
Flexible loops Hold or protect the Triosephosphate isomerase;
substrate (ligand) adenylate kinase; HIV-protease

Domain movements Induced fit, generation Hexokinase; citrate synthase;

of active site transglutaminase; cAMP-
dependent protein kinase;
adenylate kinase

Subunit communications Allostery, co-operativity Aspartate transcarbamylase;

hemoglobin
 Few examples

H57 A C
D102
S195
O H N
N HO S
O 2.7A
S
H57
D102
S195
O H N
N HO
O <2.6A

D
D
B
2
1
O2
S S
1
2
LBHB
pKa matching
 2.Electrostatic Catalysis
(Pre-organized active site)
A C
H3C R2 H3C R2
- -
+
+
R1 N S R1 N S NH2
H3C + CO2
O H3C OH HOOC COOH
HO
O

N155 B
O
O O
N
H N + H2N
E-Ser-O- E-Ser-O H E-Ser-O
(S221) (S221) (S221)
Tetrahedral intermediate
 Charge Guidance

AcCh+ O2 -
AcCh+ O 2-
+ O 2- -
AcCh+

-
+

active site active site

Acetyl cholinesterase Superoxide dismutase

AcChoEase & Gc-Lipase
Dipole one order less
in SOD
 3.Metal ion Catalysis

About 1/3rd of known enzymes use/need

metal ions in catalysis

Besides their redox properties, they

assist in catalysis through
 Charge Stabilization
A
O O
C 2+
Mn
O O O O
O C 2+ C 2+
Mn Mn
O O
C O CO2 H2C H2C
O

B
O P
O P O P
H2C
H2C H2C
2+
O Zn 2+ 2+
O Zn O Zn
HC
OH R HC HC
OH OH
R

H H C
N N

O HO H O OH
2+ 2+
Zn Zn
H+
N N N
N N N

Enzyme Enzyme
Better nucleophilicity to water

A B

O H
2+
O 2+
C Mn Mn
HO
2+ O
Zn O O
N N
N
Enzyme
Asp
Enzyme
 Charge Shielding

Nu -

O O O
O O
O P P P O
N
H2N O O O O
N

N 2+
N Mg
HO OH

Kinases and ATP: Role of Mg2+

Job Plot and stoichiometry
Structure of ATP-Me2+
O
O
P
O
O O
P
O
NH2 Me O
7
N O P
N O
N O
N
O
HO
HO
 4.General acid-base catalysis

Predominates at least one proton

transfer step in enzyme catalysis

Choice of acid-base groups

Conjugate acid-base pairs

1,3-prototropic shifts are most

common
 Concerted acid-base catalysis

A H A H
O O O

H3C O CH3 H3C O CH3 H3C O CH3

O O O
H H H H H H

B B
Acid catalysis Base catalysis Concerted Acid-Base catalysis
 Functional group Structural form Typical pKa Acts at
Acid Base (Range) pH 7.0 as
R O R O
Carboxylate 3.4 (2.0-7.0) Base
-COOH (C- OH O 3.9 (2.0-7.0)
terminal) 4.3 (2.0-7.0)
-COOH (Asp)
-COOH (Glu)
H
Imidazole (His) +
R N 6.5 (6.0-8.0) Acid or
R N
Base
N N
H H
-Amino (N- O O 7.5 (6.0-8.0) Acid
+
terminal) NH 3 NH 2
R R
+
-Amino (Lys) NH 3 NH 2 10.5 (6.0-10.5) Acid
R R

SH S
Bronsted acid-base
Sulfhydryl (Cys) 8.3 (7.5-9.0) Acid
R R groups on enzymes
Hydroxyl (Tyr) OH O 10.0 (8.5-10.5) Acid

R R

+
Guanidinium (Arg) NH 2 NH >12.0 Positive
N N
NH 2
Charge
NH 2
R R
Hydroxyl (Ser, Thr) OH O 13.5 (9.0-13.5) Acid (?)
R R

Me 2+ -H 2 O Me 2+ OH 2 Me 2+ OH 8.0 (7.0-9.0) Acid

(Mg2+ , Mn2+ , Zn 2+ etc)
Microenvironment and ionization

+ + + +
+ + + +
NH3 NH2 NH3 NH2

Normal Less basic

O OH O O O OH O O
C C C C

Normal Less acidic

O OH O O O OH O O
C C C C

Normal Less acidic

 Few examples
A A B
H O
O O O
O C C

B
C OH O
HO O
H 2C
HO
H2C
OH H:B+ H:B+

OH

O C D
R1OCH2
O Base

:O [His12]

:N
[Asp52]-COO- O OH
O
HOOC-[Glu35] NH
[His119]
O P O
+
O HN N H OR2
5.Stabilization of Transition State
O A OH B
COOH
OH OH
C H 3C H H 3C
O O O
O HO OH
D N O O
O:
HO H
OH OH HO OH HO OH
NAM HO
C H3C O N H
A c a rb o s e
O

C
E
O S ta tin
O NAG H
N NH A la S ta -C O O H
C H 3C O N H Val Va N
H
O OH O
F
NH2

N
N
O
N N NH A la
O
N
H OH
HO OH OH
HO
A d e n o s in e

OH
D
H
N
O O O
HN C C C
N N N
N N H H O H O H O
O
HO OH P ro lin e TS P y r r o le -2 -c a r b o x y la te
HO

N e b u la rin e 1 ,6 -h y d ra t e E
H H C H 2O P -OOC C H 2O P -OOC C H 2O P
N
O
HN C C OH C OH
N
N OH O OH
O HO HO HO
HO OH
HO
C H 2O P C H 2O P C H 2O P
C a rb o x y k e to n e 2 ' C a r b o x y - D - a r a b in i t o l
1 , 6 - D ih y d r o n e b u l a r i n e R ib u l o s e 1 , 5 - b is p h o s p h a t e
1 , 5 - b is p h o s p h a t e
 I think enzymes are molecules
that are complementary in structure
to the activated complexes of the
reactions that they catalyze.

Linus Pauling (1948) Nature 161:707

..ABZYMES!!
 Where does the G for TS binding
come from?

A B C D E F

--NAG-NAM-NAG-NAM-NAG-NAM--

Gbinding= -1.79 -2.94 -5.69 +2.89 -1.69 -1.69

(in kcal mol-1)
 TIM - and its lid !!!

DHAP

H OH O O OH
C H C H C N
A-H :B
OH OH
OH O
H2C P H2C H3C H2C
O P O P
METHYL GLYOXAL
ENEDIOL Phosphoglycohydroxamate
TS mimic (155 times tighter binding)

GAP
 6.Covalent catalysis

At some stage of catalysis a covalent

intermediate exists

Enzymes recruit
Electrophiles (rare on a protein!)
Nucleophiles (many on a protein!)
Examples of covalent reaction intermediates
C ovalently linked to Interm ediate E nzym e exam ples
E n zym e provides th e n ucleoph ile
S erine (-O H ) O -A cyl enz ym e A cetylcholinesterase, C hym otrypsin
C ysteine (-S H ) S -A cyl enzym e P apain, G lyceraldeh yde-3-phosphate
dehydrogenase, G lutam ate synthase
S erine (-O H ) O -P hospho enzym e A lkaline phosphatase,
P hosphoglucom utase;
P hosphodiesterase (via T hr-O H )
H istidine N 1 -P hospho enzym e G lucose-6-phosph atase, N ucleoside-
(-im idazole) bisphosphate kinase, Succinyl C oA
synthetase
T yrosine (-O H ) O -S ulfo enzym e A rylsulfate sulfotransferase
L ysine (-N H 2 ) Im ine adduct (S chiffs Fructose-1,6-bisphosphate aldolase,
base) A cetoacetate d ecarbox ylase,
T ransaldolase
L ysine (-N H 2 ) A M P -enzym e D N A ligase (N A D + )
G lutam ate (-C O O H ?) G lycosyl enzym e S ucrose phosphorylase, -
G alactosidase
E n zym e provides th e electroph ile
P yruvo yl group Im ine adduct (S chiffs H istidine decarbox ylase (bacterial)
base)
P yridoxal phosphate A ldim ine (S chiffs G lutam ate decarbox ylase, L-A lanine
base) am inotransferase
B iotin N -C arbox y-biotin A cetyl C oA carbox ylase,
T ranscarbox ylase, P yruv ate
carbox ylase
T hiazolium ring of H ydrox yeth yl T P P ; P yruvate decarbox ylase;
T hiam ine 1,2-D ihydrox yeth yl T ransketolase
p yrophosphate TPP
 Schiff base Lysine NH2
Schiff Base formation

Amine Carbonyl Carbinolamine Schiff base

H3C H3C +
CH3
H HA .. N
N O N OH H + OH-
H
H X
X X
B:

H
+
C .. C
N H N
H H

X X

Imine carbanion Enamine

 Enzyme electrophiles
derived from amino acid side chains
Amino acid residue(s) Nature of Electrophile Enzyme example
converted to modification
Lysine Pyrrolysine Pre- N
Methylamine
CH3
translational; methyltransferase
NH
dedicated O

codon usage
Polypeptide Polypeptide
N
H O

COOH
Glutamate + Tyrosine Post- HOOC HN 2
Bacterial glucose
1

PQQ (Pyrrolo- translational; 3

dehydrogenase
quinoline quinone) free cofactor 4
and methanol
HOOC N
5
O dehydrogenase
O

O
Serine Pyruvate Post- H
N Pyruvoyl enzymes
H3C Polypeptide
translational; O
like histidine
peptide decarboxylase
bound
CH2
(Alanine Post- Polypeptide Polypeptide Phenylalanine
Dehydroalanine) translational; N
H ammonia lyase
O
-Ala-Ser-Gly- - peptide and histidine
MIO- (4- bound CH2 ammonia lyase
methylideneimidazole- N
O
O
5-one) N
Polypeptide Polypeptide
 Coenzyme/ Structure Enzyme example Vitamin
Cofactor (function)
HO
Ascorbic acid O O Prolyl hydroxylase Vitamin C
HO
(Redox)
HO OH
O
Biotin Pyruvate carboxylase Biotin
HN NH
(Electrophile)
COOH
S
H2N N N
Tetrahydrofolate Serine Folic acid
N
(FH4) N hydroxymethyltransferase
OH N
H
NH Glutamate (Redox and electrophile)
O
H H
Nicotinamide CONH2 Lactate dehydrogenase Niacin
O
adenine O
(Redox)
N O P O P O N
dinucleotide O O O O
N
NH2

(NADH and HO OH
N N
HO OH
NADPH) (P)

O
Coenzyme A N O P O
Citrate synthase Pantothenic
O
(CoA) H2N
N
O O
HO
(Acyl activation; good acid
N
N N
HO
O
O P O
H H
N
SH
leaving group)
O O
O
Pyridoxal O
HC GABA transaminase Pyridoxal
OH
phosphate O P O (Electrophile)
O
(PLP) N
O
Flavin adenine HO
HO
O P O
D-Amino acid oxidase Riboflavin
dinucleotide OH
OR
(Redox)
(FAD) and N N O
Flavin NH
N
mononucleotide O

(FMN)
O
Thiamine O
P O
Transketolase Thiamine
O P O
pyrophosphate O
O (Electrophile)
(TPP) N
C
N
+
S

N NH2 H

1. The groups/ atoms relevant to chemistry are marked grey in each structure. 2. NADP+
differs from NAD+ in having a phosphate group on 2 OH (shown as (P)). 3. R= H for FMN and R=
AMP attached via its phosphate for FAD. 4. Other cofactors like lipoic acid, cyanocobalamin
(vitamin B12) and phylloquinone (vitamin K) also perform important functions but are less
Detection of reaction intermediates and
establishing their catalytic competence

Steady-state kinetics (burst phase)

Isotope exchange studies

Inference from analogs and side reactions

Direct observation and/or trapping

Stereo-chemical evidence

Catalytic competence of an intermediate

 Chymotrypsin burst kinetics
Burst Slow (Steady state)

[E] = 1.0 mg/ml

A405
[E] = 0.5 mg/ml

Time

O
O O
Fast Slow
NO2 H3C O
H3C O H3C OH
Ser-Enz OH-
O2N O (Acyl enzyme)

Enz-Ser-O- Enz-Ser-O-
(Chymotrypsin) (Chymotrypsin)
Glutamine synthetase reaction A
O
O
+ O
+
O NH3
O NH3 +
AMP O NH3
O ADP Pi
P O :NH3
O O C
O C O O C
O O
P O O
O P O NH2
O
O
L-Glutamate + ATP -Glutamyl phosphate L-Glutamine
18O PIX by glutamine synthetase B
O + AMP O + AMP O + AMP
NH3 O NH3 O NH3 O
O
P
O
P
O
P
Isotope exchange (PIX)
O O
C O
O
C O
O
O
O
C O
O
(Also in Ping-pong cases)
O O O O O
P O P O P O
O O O
O O O

Bridge 18O Rotation Nonbridge 18O

Trapping and side reaction O C

O O NH2 O
O Pi O
NH2 NH
NaBH4
O C
C
O O
H2C Pi O P O
OH
O
-Hydroxy-
-aminovalerate -Glutamyl phosphate 5-Oxoproline

Analog phosphorylation D
O O
+ O + O
H3N H3N
ATP ADP
O

H3C S NH H 3C S N P
O
O O O

L-Methionine- L-Methionine-
S-sulfoximine S-sulfoximine-P
 Nucleophile or Base?
Reaction intermediate trapping: An Example

O
Uncatalyzed
O
H3C O NO2 HO NO2 +
O
H 3C OH
H H

O
O Catalyzed Catalyzed O
Step 1 + Step 2

NO2 H3 C N NH
H3C O H3C OH
O +
HN N H HN NH
H
N-Acetyl imidazole

+
O2N O
Acetate catalyzed Hydrolysis of p-Nitrophenol

O O

H3C O NO2 H3C O NO2

O O
O H H
H3C O
H3C O

General base catalysis Nucleophilic catalysis

O O

H3C O CH3
(Acetic anhydride)

NH2
H2O
TRAP

NHCOCH3
2 CH3COOH + CH3COOH
Acetanilide
Imidazole catalyzed Hydrolysis of p-Nitrophenol

O O

H3C O NO2 H3C O NO2

O
H H
HN N
HN N

General base catalysis Nucleophilic catalysis

O
+
H3C N NH

(N-Acetyl imidazole)

NH2
H2O
TRAP

CH3COOH NHCOCH3
+ HN N + HN N
Acetanilide
 Enzyme reaction intermediates trapped by
borohydride reduction

Enzyme example Intermediate Intermediate trapped as

Fructose-1,6- Imine adduct N-Dihydroxyisopropyl derivative of active
bisphosphate (Schiffs base) site lysine
aldolase
Histidine Imine adduct N-Carboxyethyl derivative of histidine and
decarboxylase (Dehydroalanine) histamine (formed from enzyme pyruvoyl
(bacterial) group and -amino group of substrate)
Lactate racemase Lactyl thiolester Lactaldehyde (formed by reducing thioester
of enzyme cysteine)

Glutamine -Glutamyl -Hydroxy--aminovalerate (CO~OP

synthetase phosphate reduced to CH2OH)

In the first three examples the intermediate is covalently bound to the enzyme.
In the last case, the intermediate is non-covalently held at the active site.
 Stereochemical evidence
Retention of stereochemistry at C1 - Covalent glycosyl intermediate

HO H
HO-Sugar
O O-Sugar O O OH
H
C C C
H E-COO H
E-COO- E-COO-

Inversion of stereochemistry at C1 - No covalent glycosyl intermediate

HO-Sugar
O O-Sugar O H
C C
H
OH
E-COO- H OH E-COOH
Steps - Odd

Steps - Even
 Identification of catalytic residues
Besides the trapping experiments (for Nu:)

Chemical modification
pH dependence of catalysis
X-Ray and other structural data
Site directed mutations
 Chemical modification
Table 3.5 Chemical modification reagents as irreversible enzyme inhibitors
Amino acid (functional group) Commonly used reagents
Arginine (guanidinium) 2,3-Butanedione, Phenylglyoxal
Cysteine (thiol) Iodoacetamide, N-Ethylmaleimide (NEM),
4-Chloromercuribenzoate, Disulfides
Histidine (imidazole) Diethylpyrocarbonate (DEPC)

Lysine (amino) 1-Fluoro-2,4-dinitrobenzene (FDNB),

Trinitrobenzene sulfonic acid (TNBS)
Serine (hydroxyl) Diisopropylfluorophosphate (DIFP),
Phenylmethanesulfonyl fluoride (PMSF)
Tryptophan (indole) N-Bromosuccinimide (NBS)
 NEM and A. niger Glutamine synthetase
B

log kobs = log k1 + n log[ I ]

 NEM, Mn2+ and A. niger Glutamine synthetase

A B

ko k p
log = n log[ P] log K P
kp
Active site directed irreversible inhibitors

:Nu
Peptide substrate
O R
H
N H C C
C C N H C
H
O H2C O

TPCK
:Nu
H3C
O
H
N H C Cl
S C C
OO HC H2
2
 Suicide Substrates (Trojan Horse!)

Table 3.6 Suicide substrates and their target enzymes

Suicide substrate Enzyme acted upon Application

Gabaculine -Aminobutyrate transaminase Anticonvulsant
5-Fluorodeoxyuridylate Thymidylate synthase Anticancer
Clavulanic acid -Lactamase Antibiotic synergist
-Difluoromethylornithine Ornithine decarboxylase Antiprotozoal
Suicide substrate!
Origins of Enzyme Catalytic Power

Some are entropic

Proximity and
Orientation Effects

Others are enthalpic

Stabilization of
Transition State or Electrostatics
Reactive Intermediate
Different enzymes use them in
different proportions!
Enzyme Catalytic
Power (G )

General Acid-Base
Metal ion Recruitment
Catalysis

Covalent Catalysis
(Nucleophilic or
Electrophilic)
 Peptide bond hydrolysis:
Example of which enzyme uses what

O El +
N u: C

N H -X
H
Feature Serine Cysteine Aspartyl Metallo-
protease protease protease protease

Proximity and orientation

Yes Yes Yes Yes
Electrostatics
Oxyanion hole Oxyanion hole Asp-COOH to Zn2+ to polarize
polarize carbonyl
carbonyl
Acid-base catalysis
Yes Yes Yes Yes
Nucleophile used
DHS catalytic His-Cys-SH H-OH activated H-OH activated
triad; Ser-OH by Asp-COO- by Zn2+ (or Glu-
COO-)
Covalent catalysis
Acyl enzyme Acyl enzyme No No (Yes)
Transition state binding
Tetrahedral Tetrahedral Tetrahedral Tetrahedral
intermediate intermediate intermediate intermediate

Examples Chymotrypsin, Papain, Caspase, Pepsin, Renin, Carboxypeptidase

Subtilisin, Cathepsin C HIV-1 protease A, Thermolysin,
Carboxypeptidase Leucine
II aminopeptidase
 Purpose of kinetic studies -
Reaction Mechanism

1. Elementary processes

2. Rate determining step(s)

3. Insights into transition state structure