Study of Enzyme Mechanisms

• We have studied the mechanisms of peptide bond formation & hydrolysis by an enzyme

• Why study mechanisms?– Structure activity relationships → understand protein folding, etc– Understand “superfamilies”

– Design enzyme inhibitors:• Correct a metabolic imbalance• Kill an organism: Herbicides/pesticides, antibiotics

Diphtheria Toxin

• Corynebacterium diphtheriae

• ADP-ribosyltransferase• EF + NAD+ → ADP-EF +

nicotinamide• Mechanism also present

in other toxins– Pertussis, E.Coli

• Binding to EF (eukaryotes) blocks translation

Active peptide

Reaction

NN

NN

NH2

O

PPPO

N

NH2

O

OH OHOHOH

NH

O

N

NH

R

NN

NN

NH2

O

PPPO

OH OHOHOH

NH

O

N

NR

N

NH2

O

NAD+ EF - His

+

-H+

+

EF-ADP ribose

+

Potential Mechanisms?

ON

NH2

O

OH OH

O

OH OH

O

OH OH

EF

ON

NH2

O

OH OH

O

OH OH

EF

N

NH2

O

NAD+

+ +EF (Nu:)

+EF (Nu:)

+

-OR-

Active Site with NAD+ Bound (1st Step)

Hydrophobic interactions

Nu:

Testing of Mechanism

• Role of tyrosine?– Substitute with Phe → small drop in catalytic activity– Substitute with Ala → 105 drop in activity! likely responsible for substrate recognition

(hydrophobic interactions)• Other mutations show small effects• Key residues?• Glu-148 & His-21

– Mutations show large drop in catalytic activity– Glu148Ser 103 drop in activity

Plays a role in NAD+ binding

Activates incoming nucleophile

3-point binding?

NH

O

N

NH

R

O

OHOH

N

NH2

O

+

Role of Glutamic Acid in the TS?

2 possible mechanisms?

• In the absence of EF, hydrolysis of NAD+ will occur– Model the TS & understand how stabilization of TS

occurs

– Occurs via an SN2 mechanism!

H

O H

O

OHOH

N

NH2

O

+

Diphtheria Toxin as a Drug?

• Few successful inhibitors of the diphtheria toxin have been found

• Instead, the toxin’s apoptotic inducing activity has been exploited to kill Cancer cells– Active site is maintained– Alter it’s targeting ability (to cell receptor)– “Target toxin”

• Targeting polypeptide + toxic peptide (DT)

Cell receptor Cell death

Determination of Mechanism?

• How do we elucidate a biological pathway or an enzyme’s mechanism?

• Biological Methods – genetic engineering– Construction of mutants

• Chemical Methods– Construct analogues (recall the use of fluorine in tRNA)– Photochemical methods– Isotopes (stable & radioactive)

• OR can use a combination of both methods!

Isotopes

• Atoms of the same element having different numbers of neutrons & different masses– e.g. 1H, 2H, 3H & 12C, 13C, 14C

• Can be used as “markers” → exploit a unique property of isotope & detect using analytical techniques– Radioactivity– NMR activity– Different mass (mass spec.)***

• Markers can:– Elucidate a biosynthetic pathway– Provide mechansitic (transition state) information

How?

?*

Grow organismIsolate metabolite & look for marker

**

“feed” the labelled compound to the organism

Early Days - Radioisotopes

• Common “markers”:– 14C (t1/2 = 5700 y, Nat. Abund. = trace)

– 3H (t1/2 = 12 y, Nat. Abund. = 0%)

– 32P (t1/2 = 14 d, Nat. Abund = 0%)

• Once metabolite is isolated, radioactivity (decay) is detected

• Problems– Where is the isotope (marker)?

• Harsh degradation methods must be employed can take weeks

– Safety

– Availability of precursor

O-Na+

O

O

OH

OH

?

For example:

• In the 1950’s, Birch administered sodium acetate that was carbon-14 labelled to a Penicillium organism:

• Using harsh degradation methods, he was able to establish how sodium acetate was used to synthesize 6-MSA

O

OH

OH OH

CO2

6-MSA

Stable Isotopes

• With the development of pulsed NMR came the use of stable isotopes gain information on connectivity

• Mass spectrometry can be used little information location of isotope

• Commonly used: 2H, 13C, 18O & 15N• Carbon-13

– NMR active (I = ½)– Nat. abundance 1.1%– Many compounds are commercially available– Used to study the fate of carbon (i.e., C-C bonds formed &

bonds broken)

• Deuterium– NMR active (I = 1)– Nat. Abundance 0.015%

– Commercially available (i.e. D2O) & cheap!

– For the study of the fate or source of hydrogen • E.g. Which proton is deprotonated? Is a given proton from H2O or

another molecule?

• 18O and 15N– Employed to study the fate of oxygen and nitrogen – i.e., amino acids; Did oxygen come from water or oxygen?

Precursors (“what to feed”)

• Choice of isotope & compound to feed depend on pathway

• i.e.,– Sodium acetate is an intermediate in many biochemical

pathways

• Some knowledge of the pathway helps, but it is not necessary use knowledge of other pathways

• Examples

O-Na+

O OH

H2O

O2

Examples:

OHO-Na+

O

• Other possibilities:– Neighboring carbon-13 labels 13C-13C (coupled doublets)– No change in signal intensity label did not incorporate into

metabolite!

• Deuterium?– Can use 2H NMR– Don’t need to worry about “background” deuterium any

deuterium signal seen, must come from your precursor

A Look Back at 6-MSA

CD3

O

OH

OH

D

DCD3 O

O

Proposed Pathway:

CD3

O

OH

OH

D

D

CD3 O

O

O

O

enzyme

CD3

ODD

D

D

O

O

O

CD3

O

Mechanisms

• Isotopes (stable and/or radio) can also be employed elucidate a mechanism (transition state)

• Bases on the principle that there is a change in reactivity due to isotopic substitution

• How? • Kinetic isotope effects (KIEs):

– Can probe transition states directly → useful for understanding cataylic processes

KIE = lightk / heavyk

i.e.,

Why?• (An in-depth look at these principles is beyond the scope

of this course)• Recall vibration model of a bond:

CH3OH CH3O- H+

CH3OD CH3O- D+

+

+

lightk

heavyk

r

Force constant, F

• Total energy is proportional to the frequency of vibration– Related to force constant (unique to “spring”)– Related to mass change mass, change frequency

• Recall, we use the same model for IR spectroscopye.g. C-H (stretch) = 2700 – 3300 cm-1

C-D (stretch) = 2000 – 2400 cm-1

changing the mass can affect the rate at which a bond is cleaved or formed → reaction rate!

CHCl3

CDCl3

• (Can also use quantum mechanical methods)• Primary KIE

– The effect is occurring on atom undergoing substitution

i.e.

A KIE can provide info on the change in the environment (vibrational) in going from reactant to TS

C13 X

H

H H

C13

H

HH

Nu X+Nu: +

Measuring Isotope Effects

Competitive KIEs• Measure the rate constants• Labelled & unlabelled reactants are combined in a single

reaction mixture → allowed to react as competitive substrates (enzymatic or non-enzymatic)

• Measure the light/heavy at different times

• End up with a KIEobs

• If KIE = 0, then no isotope effect atom is not near reacting site

• How?• Measure isotopic ratios using:

– Mass spectrometry– Radioactivity (very efficient)

• 3H, 14C & 32P– Can also use NMR (not trivial!)

Application

• Recall the hydrolysis of NAD+:

• TS determined by KIEs:• Isotopes used to

determine that both Nu: and nicotinamide ring are both in the TS

H

O H

O

OHOH

N

NH2

O

+