1 / 28

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

arleen
Télécharger la présentation

Study of Enzyme Mechanisms

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related