Key features of cysteine proteinase mechanism defined by kinetic, spectroscopic and protein engineering techniques

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Project objective

Research objectives and content The cysteine proteinase superfamily which includes enzymes of biological and medical importance is a source of natural structural variants of value in the investigation of mechanism. Recent work in the QMW Laboratory has produced definitive evidence that the ion-pair state (Cys25)_S-/(HislS9)-/Im+ (C-H+) is formed across pKa valueS 2.5-3.3 and is fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation at higher pH values with a pKa value close to 4. Experiments on Asp158Asn mutants have ruled out Asp158, the nearest carboxylate to the ion-pair, as the electrostatic switch. Electrostatics potential calculations suggest that two electrostatic clusters, centred on Glu50 and Glu 135 respectively, are prime candidates to contribute the carboxylate switch. The research objective is to generate a range of mutants of papain and caricain and to characterize the active centre chemistry and catalytic characteristics by stopped-flow kinetics using specially designed and synthesized time-dependent inhibitors as reactivity probes. Crystal structures of the key mutants will be determined in collaboration with IFR, Reading, UK. The ionization characteristics of the catalytic site histidine sidechains will be determined by NMR spectroscopy. The project will produce a detailed description of cysteine proteinase mechanism and will contribute substantially to our understanding of the roles of electrostatic effects and key binding interactions in catalytic site chemistry. Training content (objective, benefit and expected impact) Studies on the natural cysteine proteinase variants and the key mutants will properly define for the first time the catalytic mechanism of this class of enzymes. Training will be provided in the study of catalytic mechanism by rapid kinetics, protein engineering and computational techniques. The acquisition of these techniques will complement my existing experience and expertise in the fields of organic synthesis, immunology and protein chemistry. Links with industry / industrial relevance (22)

Original text from CORDIS.

Participants

• QUEEN MARY AND WESTFIELD COLLEGE · LONDONCoordinatorUnited Kingdom
• Not availableCity levelItaly

Links

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