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

Potential Mechanisms? -OR-

Active Site with NAD + Bound (1 st Step) Hydrophobic interactions Nu:

Testing of Mechanism Role of tyrosine? –Substitute with Phe → small drop in catalytic activity –Substitute with Ala → 10 5 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 10 3 drop in activity

Plays a role in NAD + binding Activates incoming nucleophile 3-point binding?

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 S N 2 mechanism!

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 receptorCell 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. 1 H, 2 H, 3 H & 12 C, 13 C, 14 C 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 organism Isolate metabolite & look for marker * * “feed” the labelled compound to the organism

Early Days - Radioisotopes Common “markers”: – 14 C (t 1/2 = 5700 y, Nat. Abund. = trace) – 3 H (t 1/2 = 12 y, Nat. Abund. = 0%) – 32 P (t 1/2 = 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

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

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: 2 H, 13 C, 18 O & 15 N 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. D 2 O) & cheap! –For the study of the fate or source of hydrogen E.g. Which proton is deprotonated? Is a given proton from H 2 O or another molecule? 18 O and 15 N –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

Examples:

Other possibilities: –Neighboring carbon-13 labels  13 C- 13 C (coupled doublets) –No change in signal intensity  label did not incorporate into metabolite! Deuterium? –Can use 2 H NMR –Don’t need to worry about “background” deuterium  any deuterium signal seen, must come from your precursor

A Look Back at 6-MSA Proposed Pathway:

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 = light k / heavy k

i.e., Why? (An in-depth look at these principles is beyond the scope of this course) Recall vibration model of a bond: 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 spectroscopy e.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! CHCl 3 CDCl 3

(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

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

How? Measure isotopic ratios using: –Mass spectrometry –Radioactivity (very efficient) 3 H, 14 C & 32 P –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