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

  13. Early Days - Radioisotopes

  14. Stable Isotopes • Commonly used: 2H, 13C, 18O & 15N

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