BIOC 3007
Advanced enzymology

X-ray crystallography of proteins. Kinetic analysis of multi-substrate enzymes, effects of pH and allosteric regulation

PLEASE NOTE:  This unit will cease to run after the completion of the current academic year and will not be continued in the academic year 2003-2004

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Semester
Unit Director: Dr. B. Hayden
1-6 Lectures: Dr C. Sharkey (X-ray crystallography of proteins)
7-9 Lectures: Professor J.P.G. Malthouse (Effects of pH) (3 lectures, JPGM)
10-15 Lectures: Professor P.C. Engel (Kinetic mechanisms of multisubstrate enzymes)
16-22 Lectures: Dr. B. Hayden (Product inhibition, isotope exchange at equilibrium, pre-steady state kinetics Allosteric enzymes)


Practical classes
Molecular modelling 1 (dry, CS)
Molecular modelling 2 (dry, CS)
pH effects 1 (data analysis, JPGM)
Inhibition of ADH by pyrazole (wet, SGM)
Hill plot (data analysis, SGM)


Lecture content

1. Importance of structure to understanding mechanism. Introduction to diffraction. Crystals. Crystal growth.
2. Recovering info from diffraction patterns. Coordinate sytems. Wave equations and complicated periodic functions. Structure factors. Electron density.
3. Relationship between structure factor and electron density. Model building. Bragg's Law. Unit cells. Miller indices. The reciprocal lattice
4. Fourier Series and Fourier Transforms. Representation of structure factors and electron density
5. Phases. Complex number representation of structure factors. Heavy atom replacement. Obtaining phases.
6. Phases (continued). Patterson synthesis methods.
7. Acid-Base catalysis. General versus Specific Acid-Base catalysis. Acid-Base catalysis in the mechanism of action of serine proteases. Effect of electrostatic interactions on pKa values.
8. pH effects on simple chemical reactions. Singly and doubly ionising systems. Estimation of pKa values.
9. Overlapping pKa values. Microscopic and macroscopic pKa values. pH dependence of binding, kcat and kcat/Km. Kinetic pKa values. pH dependence of kcat and kcat/Km for papain and chymotrypsin catalysed reactions.

Kinetic mechanisms of multisubstrate enzymes (6 lectures, PCE)

10. Why multi-substrate kinetics? Consider the EC classifications which underline how few one-substrate reactions there are. Detour to consider the special issue (in terms of terminology) of cofactors vs. substrates/coenzymes/prosthetic groups etc. - functional definitions. The various roles of metal ions in enzyme reactions.
11. Various types of mechanism - is there a central complex? random/compulsory. Derivation of two substrate compulsory-order steady-state mechanism equation
12. Dalziel Ø notation
Ways in which Ø parameters or ratios can be compared with:
• Equilibrium constants
• Kinetic data for reverse reaction
• Kinetic data for alternative substrates
• Directly measured binding constants
• Individual rate constants from rapid kinetics

13. Collapse to TC mechanism. Exemplified with real data for Yeast ADH
14. Random Order:
• Non-linearity
• Special case of rapid-equilibrium
• all Kd's for comparison

15. Ping Pong Mechanism : Special methods applicable - direct observation of partial reactions and/or measurement of isotope exchange in absence of one substrate
16. Product inhibition of 2-substrate enzymes. Isotope exchange at equilibrium as a method to distinguish between ordered mechanisms for 2-substrate enzymes, with malate dehydrogenase as an example.
17. Pre-steady state kinetics. Methods for direct measurement of enzyme rate constants; continuous and stopped flow spectrophotometry; relaxation techniques (temperature- and pH-jump).
18. Introduction to allosteric control of enzyme activity. Experimental observations on pyrimidine biosynthesis and amino acid metabolism in E. coli that led to Monod and Koshland models. The Hill equation; derivation for haemoglobin; application to enzymes
19. Monod model for positive homotropic cooperativity. Qualitative treatment. Derivation of equation to describe fractional saturation of two-subunit enzyme by substrate. Applications and limitations. K and V systems
20. Koshland model. Qualitative treatment to describe different equilibria in 2-subunit system. Stability constants for different forms of dimer. Application. Tests for the models.
21. Description of aspartate transcarbamoylase; reaction catalysed; effects of substrate and effector concentrations; structure
22. Tests on ATCase for Monod and Koshland models; mutagenesis to test important interactions between subunits

Practical classses

• Molecular modelling 2 sessions (CS)
  
• pH effects (data analysis, JPGM)
  
• A critical comparison of the different methods for the estimation of pKa values e.g. by visual inspection of sigmoidal pH data, by using of reciprocal plots or log plots. The use of log plots to estimate pKa values from singly and doubly ionising systems.
  
• Inhibition of ADH by pyrazole (wet, SGM)
  
• Inhibition of yeast alcohol dehydrogenase. Practical exercise to examine the effects of pyrazole on the initial rate of formation of NADH; determination of apparent K_m and apparent V_max; secondary plot to calculate K_i.
  
• Hill plot (data analysis, SGM)
  
• The binding of ligands to enzymes - the Hill coefficient; theory and data analysis