enzymology: kinetics and mechanismsflashman.chem.ox.ac.uk/docs/flashman_enzymology_2nd_year... ·...

UNIVERSITY OF OXFORD

DEPARTMENT OF CHEMISTRY

Enzymology:

Kinetics and Mechanisms

Dr Emily Flashman

http://flashman.chem.ox.ac.uk

4 Lectures – Trinity Term 2015

Lecture 1: Enzyme kinetics (Michaelis Menten)

Case study – chymotrypsin

Kinetics of multi-substrate reactions

Lecture 2: Enzyme mechanisms

Inverting and retaining glycosidases

Case study – lysozyme

Hydroxylase enzymes – use of cofactors

Case study 2 – phenylalanine hydroxylase (including

pre-steady state kinetics)

Lecture 3: Enzyme inhibition

Classes of enzyme inhibitor and mechanisms

Effects on enzyme kinetics

Use of kinetic data to identify potent inhibitors.

Lecture 4: Protease Inhibition

Case study – HIV aspartic protease

Mechanism and inhibitor development

Other proteases

Topics to be Covered: Enzymology: Lecture 4

Enzymology: Lecture 1

• No text books are essential but the following are useful:

What you need:

• Selected journal articles to supplement understanding

Enzymes: A Recap Enzymology: Lecture 1

Enzymes are protein-based catalysts – they facilitate chemistry

Bringing enzyme and substrate(s) together in a favourable

conformation to promote a specific reaction.


Enzymes are important – enable the chemistry of life

Understand and manipulate enzyme activity • identify and test pharmaceutical targets • understand human (and other) biology • biocatalysis • many others, e.g. industrial use, asymmetric resolutions, bioremediation

Enzymes: A Recap

Enzymes are biological catalysts:

Enzymology Lecture 1

Increase the rate at which a reaction reaches equilibrium

Stabilise the transition state of a reaction relative to the uncatalysed reaction

Enzymes are finely tuned for specificity in substrate binding and optimal arrangement of catalytic groups

Enzymes: A Recap

Enzyme Kinetics Enzymology: Lecture 1

Enzymes enhance the rate of a reaction to be compatible with the needs of the organism Enzymes: perform a variety of reactions via a number of mechanisms e.g. single substrate, multiple substrates, defined binding order The study of enzyme kinetics allows the determination of enzyme mechanism i.e. whether they can work at maximal efficiency under different conditions We can understand how they work Study the RATE of enzyme reaction under defined conditions

Measure as a function of substrate consumption/ product formation

Enzymology: Lecture 1 Enzyme Kinetics: Michaelis-Menten Model

E = enzyme S = substrate P = product

For an enzyme reaction, known quantities are rate (v, [P] with time), [S]0 and [E]0. These do not inform on mechanism, nor allow comparison between enzymes/conditions.

v = rate of enzyme activity, measured as product formation/time

kcat = 1st order rate constant (units s-1)

A measure of how active an enzyme is in reacting with a substrate once it is bound as an ES complex

MK

kcat= 2nd order rate constant (units s-1 M-1)

A measure of the catalytic effectiveness of an enzyme in dilute solution, that includes the specific binding of the substrate to the enzyme.

E + S ES E + P

k1

k2

k3

MKk

kk

1

32= Michaelis constant (units M-1)

A measure of the stability of the ES complex


Measure enzyme activity in terms of rate and [S] Fit data to hyperbolic curve with Michaelis-Menten equation to reveal kcat and KM

M

max[S]

[S]

KVV

Where:

MKk

kk

1

32


When S is in excess, only the 2nd step (where mechanism occurs) is limiting.

Principle features of enzyme-catalysed reactions: 1. For a given [S]0, v is proportional to

[E]0.

2. For a given [E]0 and low [S]0, v is proportional to [S]0.

3. For a given [E]0 and high [S]0, v is

independent of [S]0, reaching Vmax.


Reaction must be in the steady state, i.e. before equilibrium is reached. No product reverts to substrate Substrate is in excess over Product and Enzyme


Michaelis-Menton Model

E + S ES E + P

k1

k2

k3

[ES]

[E][S]MK

]ES[3kv Rate of product formation requires us to know [ES]

Can’t know [ES], so need to define in terms we can input

0][k][k]S][[kdt

][d321 ESESE

ESUnder steady state approximation

]S][E[ [ES]32

1

kk

kMK

k

kk

1

32


M

T [ES])[S]]([E[ES]

K

M

T[S]

[S]][E[ES]

K

[ES] [E] ][ET [E]T = total enzyme (known)

[ES]

[E][S]MK

M

T [ES][S]][S][E[ES]

K

][SE][[ES][S][ES] TMK

][SE][[S])[ES]( TMK

Know that v=k3[ES]

M

T3

[S]

[S]][E

Kkv

Solve for [ES]:

Know that Vmax=k3[ET]

M

max

[S]

[S]

KVv Michaelis-Menten equation


M

max

[S]

[S]

KVv

A way to ascertain useful information (KM and Vmax) in measurable terms (v and [S]).

If k2 >> k3 then 1

2M

k

kK

In this case and ONLY IN THIS CASE:

KM = Dissociation constant of the ES complex

and as such as a measure of the ‘strength’ of the ES complex.

High KM = weak binding, low KM = strong binding.

MKk

kk

1

32


Taking reciprocals:

M

max[S]

[S]

KVV

[S]

111

max

M

max

V

K

VV

Plot vs

A Lineweaver-Burke plot V

1

][

1

S:

Lineweaver-Burke plot gives best precision for estimates of KM and Vmax

Weakness of Lineweaver-Burke plot is that data is ‘weighted’ differently at high and low [S], so it can be inaccurate.

Enzymology: Lecture 1 Determination of Vmax and KM

max

][V

S

vKv M

Eadie Hoffstee plot – v (dependent variable) used on both axes, errors multiplied

Enzymology: Lecture 4 Determination of Vmax and KM

v

v/[S]

-KM

Vmax/KM

Vmax

maxmax /][//][ VSVKvS M

Hanes plot – [S] used on both axes, experimental errors multiplied

[S]/v

[S] -KM

KM/Vmax

1/Vmax

1. Most enzymes do NOT operate under saturating conditions.

Therefore criterion for measuring efficiency

MK

kcat (2nd order rate constant)

Rate dependent on [E] and [S]

Ultimate limit set by

i.e. finally comes down to k1

2. Both and are useful for comparing different enzymes / substrates.

1

32M

k

kkK

catk

MK

kcat


Enzymology: Lecture 1 Case Study: Chymotrypsin

In isolation: Amide bond highly stable, acid or base needed for hydrolysis

Serine proteases cleave peptide (amide) bonds under physiological conditions:

Nucleophile

Acid/base catalyst

Oxyanion hole

Proteases

Case Study: Chymotrypsin Enzymology: Lecture 1

Enzymology: Lecture 1 Case Study: Chymotrypsin


Tetrahedral intermediates stabilised via oxyanion hole

Case Study: Chymotrypsin

Evidence for 2-phase reaction:

• Chymotrypsin can also catalyse the near-equivalent ester hydrolysis reaction • Chymotrypsin-catalysed hydrolysis of p-Nitrophenylacetate

Chymotrypsin – Evidence for mechanism Enzymology: Lecture 1

• Rate of burst phase dependent on [substrate] • Rate of steady state phase independent of

[substrate] • 2 phase reaction: rapid formation of acyl-

enzyme intermediate then slower hydrolysis

yellow

Identification of Ser-195 role in catalysis

• In the case of chymotrypsin phenylmethylsulphonyl fluoride was shown to be a potent inhibitor. • After digestion of the inhibited enzyme with a protease, the inhibitor was shown to be linked to Ser-195.

Digestion Analysis


Nerve poisons act in this way

Chymotrypsin – Evidence for mechanism

Identification of His-57 role in catalysis

• tosyl-l-phenylalanine chloromethyl ketone (TPCK) is a substrate analogue that

reacts to form a stable covalent bond (Trojan horse)


• This reaction can be prevented by competitive substrate analogues, 8M urea or by blocking active site

Chymotrypsin – Evidence for mechanism

Site-Directed Mutagenesis is a technique to modify targetted amino acid residues in a protein to examine their function

e.g. Examining the role of Asp-102 of chymotrypsin

•Mutate Asp-102 to Asn-102

Result:

KM = similar to wildtype chymotrypsin

kcat = decreased

Therefore, Asp-102 is probably not involved in substrate binding, but important in catalysis.

Enzymology: Lecture 1 Chymotrypsin: Kinetic evidence for mechanism

WT enzyme

Asp102Asn

e.g. Examining the role of Ser-195 of chymotrypsin

•Mutate Ser-195 to Ala-195

Result:

KM = remains the same

kcat = significantly reduced

Therefore, formation of ES link is NOT essential for activity

Ser/Asp/His triple Ala mutant still shows >1000 fold rate acceleration – why?


WT enzyme

Ser195Ala

Chymotrypsin: Kinetic evidence for mechanism

Kinetic constants for the hydrolysis of:

• N-Acetyl-L-Tyrosine-Glycinamide (A)

• N-Acetyl-L-Tyrosine Ethyl Ester (B) at pH 7.9, 25C

Amide (A) Ester (B) B / A

kcat (s-1) 0.50 193 390

KM (M) 0.023 0.0007 0.03

kcat / KM (M-1s-1) 22 280,000 12,700

1. Under saturating conditions (kcat), ester hydrolysis 390x faster than amide hydrolysis

2. Under low [S] conditions (kcat / KM), ester hydrolysis 12,700x times faster than amide

hydrolysis.

• Differences are due to differences in KM - enzyme becomes saturated at a lower concentration

of the ester.

• Not necessarily due to more stable ES complex….

CH3CONHX

O

OH

A, X=

B, X = OEt

HNNH2

O


Remember, chymotrypsin can catalyse hydrolysis of amides or esters


1. k3/cat for ester > kcat for amide. (reasonable as esters are much more susceptible to chemical hydrolysis than amides).

2. For esters k2 NOT >> kcat i.e. KM does NOT represent the dissociation constant of the ES complex.


Kinetic constants for the hydrolysis of N-Acetyl-L-Tryptophan derivatives: Ac-(L-Trp)-X

X kcat (sec-1) KM x 103 (M)

OEt 27 0.097

OMe 28 0.095

O-p-C6H4NO2 31 0.002

NH2 0.026 7.3

1. Identical kcat values for the three esters For ester hydrolysis, breakdown of common intermediate (acyl-enzyme) is rate-limiting. Formation of the acyl enzyme intermediate is rapid and the ES complex exists predominately in the form of the acyl-enzyme.

2. kcat value is much lower for the amide For amide hydrolysis, formation of the acyl-enzyme is rate-limiting

CH3CONHX

O

NH

O

N+

O–

O

Ac-(L-Trp)-X


E-OH + RCOX E-OH.RCOX

E-O-COR

E-OH + RCO2H

k1

k2

k3 HX

H2O

(Ser-OH)

(Acyl-Enzyme)

k4

r.l.s foramides

r.l.s foresters

43

4

1

32

Mkk

k

k

kkK

43

43cat

kk

kkk

Esters are good acylating agents, hence k3 > k4 and kcat = k4, BREAKDOWN of the acyl-enzyme is rate-limiting and the same for all the esters.

Amides are relatively poor acylating agents, hence the acylation step is much slower, k4 > k3, hence kcat = k3 and the ACYLATION step is rate limiting

Note: for amides k2 >> k3 and k3 << k4.

Therefore KM = A binding constant for the ES complex

For esters k2 NOT >> k3 , therefore KM binding constant for the ES complex

1

2

k

k



Ionisable residue is probably involved in catalysis.

Protonated imidazole group of His-57 has pKa of 7

A terminal amino group has a pKa value 8.5

For amides,

Therefore pH dependence of reflects substrate binding. 1

2M

k

kK

M

1

K


N

N O

O H

H O

O O -

NH 3 +

Asp-102

His-57 Ser-195

Ile-16

Asp-194

pH 10 inactive

N

N O

O H

H O NH 3 +

Asp-102

His-57 Ser-195

Ile-16

Asp-194

pH 7 active

O

O -

blocked pocket

open pocket

NH2 NH3+


Remember hexokinase from last year? Catalyses nucleophilic attack of C6-OH on ATP

Glucose + ATP Glucose-6-phosphate Hexokinase

Mg++

E + S ES E + P

k1

k2

k3

Kinetics of Multi-Substrate Reactions


Sequential Reaction Mechanisms

Glucose + ATP Glucose-6-phosphate

Hexokinase

Mg++


All substrates must bind to the enzyme before the reaction can proceed


Ping Pong Reaction Mechanisms

Polypeptide(n=12) + H2O 2 x Polypeptides(n=6)

Chymotrypsin

Products are released in step-wise fashion; after initial reaction with S1, a new stable form of the enzyme is generated. For chymotrypsin this is E’, the acyl-enzyme intermediate. S1 = polypeptide, S2 = H2O.


For detailed derivation of rate kinetics in each case, refer to ‘Physical Chemistry for the Life Sciences’ (Atkins and de Paula), Section 8.2.

Enzymology: Lecture 1 Kinetics of Multi-Substrate Reactions

1/v

1/[S1]

Increasing [S2]

Sequential Reaction

1/v

1/[S1]

Increasing [S2]

Ping-Pong Reaction

Experiment to determine mechanism of 2-substrate reaction Fix [S2], plot rate dependence on [S1]; repeat at varied [S2] For sequential mechanism, slope of plot depends on S2 For ping-pong mechanism, slope of plot independent of S2

Note, typical ping-pong mechanism graph not seen for chymotrypsin because S2 is H2O, concentration not variable.

]S[

1

V

]S/[KKK

V

]S/[K1

v 1

2M1M21M

max

212M

max

12

1

]S[

1

V

K

V

]S/[K1

v

1

1max2

1M

max2

22M

2

enzymology: kinetics and mechanismsflashman.chem.ox.ac.uk/docs/flashman_enzymology_2nd_year... ·...

Documents