Professor Keith Brocklehurst

Research

Research Interests:

Mechanism-targeted kinetic studies on enzymes and catalytic antibodies

Structure-function and mechanistic studies on enzymes and catalytic antibodies, principally by stopped-flow kinetic, protein chemical, protein engineering, and computational techniques; mathematical analysis of kinetic phenomena, particularly pH-dependence and optimal kinetic characteristics of catalysis.

Particular emphasis on natural and engineered variation in the cysteine proteinase superfamily and the design, synthesis and use of two-protonic-state time-dependent inhibitors as reactivity probes and thionoester substrates as detectors of covalent intermediates and conformational isomers; dependence of catalytic site chemistry on molecular recognition interactions, electrostatic effects and active centre solvation-desolvation phenomena; the extent to which crystal structures predict kinetic and mechanistic characteristics.

Publications

• Brocklehurst, K., Gul, S. and Pickersgill, R.W. (2009) Substrate recognition. In: Amino acids, peptides and proteins in organic chemistry (ed. A.B. Hughes) Wiley-VCH, in press.
• Gul,S., Hussain,S., Thomas,M.P., Resmini,M., Verma,C.S., Thomas,E.W., and Brocklehurst,K. (2008) Generation of Nucleophilic Character in the Cys25/His159 Ion Pair of Papain Involves Trp177 but Not Asp158 Biochemistry, 47: 2025-35.
• Fries M, Ihrig J, Brocklehurst K, Shevchik VE, Pickersgill RW. (2007) Molecular basis of the activity of the phytopathogen pectin methylesterase. EMBO J. 26(17):3879-87.
• Frank S, Deery E, Brindley AA, Leech HK, Lawrence A, Heathcote P, Schubert HL, Brocklehurst K, Rigby SE, Warren MJ, Pickersgill RW. (2007) Elucidation of substrate specificity in the cobalamin (vitamin B12) biosynthetic methyltransferases. Structure and function of the C20 methyltransferase (CbiL) from Methanothermobacter thermautotrophicus. J Biol Chem. 282(33):23957-69.
• Boucher G, Said B, Ostler EL, Resmini M, Brocklehurst K, Gallacher G. (2007) Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody. Biochem J. 401(3):721-6.
• Gul S., Mellor G.W., Thomas E.W. and Brocklehurst K. (2006) Temperature-dependences of the kinetics of the reactions of papain and actinidin with a series of reactivity probes differing in key molecular recognition features. Biochem. J. 396, 17 -21.
• Graham C.E., Brocklehurst K., Pickersgill R.W. and Warren M.J. (2006) Characterization of retinaldehyde dehydrogenase 3. Biochem. J. 394, 67-75.
• Sonkaria S., Boucher J., Florez-Alvarez J., Said B., Hussain S., Ostler E.L., Gul S., Thomas E.W., Resmini M., Gallacher G. and Brocklehurst K. (2004) Evidence for 'lock and key' character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: differences in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes. Biochem. J. 381,125-130.
• Reid J.D., Hussain S., Bailey T.S.F., Sonkaria S., Sreedharan S.K., Thomas E.W., Resmini M. and Brocklehurst K. (2004) Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation. Biochem. J.378, 699-703.
• Brocklehurst K., Courey A.J., Gul S., Lin S-H and Moritz R.L. (2004)Affinity and immunoaffinity chromatography. In: Purifying Proteins for Proteomics (ed. R.J. Simpson) Cold Spring Harbor Laboratory Press, New York, pp 221-273.
• Resmini M., Ostler E.L., Brocklehurst K. and Gallacher G. (2004) Polyclonal catalytic antibodies. In: Catalytic Antibodies (ed. E. Keinan) UCH-Wiley, Chapter 17, pp 468-488.
• Watts A.B. and Brocklehurst K. (2004) Streptopain. In: Handbook of Proteolytic Enzymes (eds. A.J. Barrett, N.D. Rawlings and J.F. Woessner) 2nd Edition, Academic Press, London pp 1245-1249.
• Watts A.B. and Brocklehurst K. (2004) Actinidain. In: Handbook of Proteolytic Enzymes (eds. A.J. Barrett, N.D. Rawlings and J.F. Woessner) 2nd Edition, Academic Press, London, pp 1143-1146.
• Gul S., Sonkaria S., Pinitglang S., Florez-Álvarez J., Hussain S., Thomas E.W., Ostler E.L., Resmini M., Gallacher G. and Brocklehurst K. (2003) Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving group determinant: unequivocal evidence for the ‘flexibility’ hypothesis. Biochem. J. 376, 813-821.
• Hussain S., Pinitglang S., Bailey T.S.F., Reid J.D., Noble M.A., Resmini M., Thomas E.W., Greaves R.B., Verma C.S. and Brocklehurst K. (2003) Variation in the pH-dependent pre-steady state and steady state kinetic characteristics of cysteine proteinase catalytic mechanism: evidence for electrostatic modulation of catalytic site function by the neighbouring carboxylate anion. Biochem. J. 372, 735-746.
• Hussain S., Khan A. and Brocklehurst K. (2002). A review of developments in the study of cysteine proteinase mechanism: opportunities for investigation of electrostatic effects and dynamic aspects of molecular recognition in enzyme chemistry. Recent Res. Devel. Biochem. 2, 653-677.
• Ostler E.L, Dean C.J., Barber N., Valeri M., James S., Resmini M., Boucher G., Romanov N., Brocklehurst K. and Gallacher G. (2002). A rat monoclonal antibody that catalyses the hydrolysis of hapten-related nitrophenyl carbonates and a nitrophenyl B-lactam. Biochemical and Biophysical Research Communications 299, 273-276.
• Ostler E.L, Resmini M., Brocklehurst K. and Gallacher G. (2002). Polyclonal catalytic antibodies. Journal of Immunol. Methods 269, 111-124.
• Ostler E.L, Resmini M., Boucher G., Romanov N., Brocklehurst K. and Gallacher G. (2002). Polyclonal antibody catalysed hydrolysis of a B-lactam. Chemical Communications 226-227.
• Florez-Alvarez J., Brocklehurst K., Gallacher G. and Resmini M. (2002). Synthesis of an amino alcohol hapten for the generation of catalytic antibodies. Tetrahedron Letters 43, 171-174.
• Brocklehurst K. (2002). pH-stat Assays. In: Enzyme Assays: A Practical Approach (eds. R. Eisenthal and M. J. Danson), 2nd edition, 157-170. Oxford University Press.
• Reid J.D., Hussain S., Sreedharan S.K., Bailey T.S.F., Pinitglang S., Thomas E.W., Verma C.S. and Brocklehurst K. (2001). Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations. Biochem. J. 357, 343-352.
• Brocklehurst K., Resmini M. and Topham C.M (2001). Kinetic and titration methods for determination of active-site contents of enzyme and catalytic antibody preparations. Methods 24, 153-167.
• Krell T., McLean J., Boam D. J., Cooper A., Resmini M., Brocklehurst K., Kelly S.M., Price N.C., Lapthorn A.J. and Coggins J.R. (2001). Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine. Protein Science 10, 1137-1149.