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Study of Enzyme Mechanisms

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

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Study of Enzyme Mechanisms

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  1. 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

  2. Diphtheria Toxin Active peptide • Corynebacterium diphtheriae • ADP-ribosyltransferase • EF + NAD+→ ADP-EF + nicotinamide • Mechanism also present in other toxins • Pertussis, E.Coli • Binding to EF (eukaryotes) blocks translation

  3. Reaction

  4. Potential Mechanisms? -OR-

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

  6. 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

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

  8. Role of Glutamic Acid in the TS?

  9. 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!

  10. 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

  11. 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!

  12. 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

  13. How? * ? “feed” the labelled compound to the organism Grow organism Isolate metabolite & look for marker * *

  14. 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

  15. For example: • In the 1950’s, Birch administered sodium acetate that was carbon-14 labelled to a Penicilliumorganism: • Using harsh degradation methods, he was able to establish how sodium acetate was used to synthesize 6-MSA

  16. 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)

  17. 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?

  18. 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

  19. Examples:

  20. 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

  21. A Look Back at 6-MSA Proposed Pathway:

  22. 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

  23. i.e., Why? • (An in-depth look at these principles is beyond the scope of this course) • Recall vibration model of a bond: Force constant, F r

  24. 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! CHCl3 CDCl3

  25. (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

  26. 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

  27. How? • Measure isotopic ratios using: • Mass spectrometry • Radioactivity (very efficient) • 3H, 14C & 32P • Can also use NMR (not trivial!)

  28. 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

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