Dissecting catalytic effects in the active site of ferrochelatase.

Context of the research Enzyme catalysis is an enthralling and significant problem with far-reaching consequences for biotechnology and medicine. Deeper insights into enzyme action are needed if we are to turn the massive increase in biological information, arising from structural biology and genome projects, into real biological and molecular understanding. Research programmes directed at fundamental enzymology are particularly timely as it is becoming clear that modern techniques in structural biology and site-directed mutagenesis coupled to improvements in computing and kinetic instrumentation have prepared the ground for a renaissance in mechanistic biochemistry. Aims and objectives I intend to investigate the fundamental basis of enzyme catalysis using the haem biosynthetic enzyme ferrochelatase. Earlier crystallographic and spectroscopic work shows that ferrochelatase binds and bends the porphyrin substrate. Chemical experience suggests that this helps the enzyme deprotonate the substrate and to insert the metal ion. I aim to use a combination of transient kinetics, structural variation and spectroscopy to explore the interplay between substrate distortion and general-base catalysis; strategies used across all enzyme families. Potential applications and benefits We will all benefit from a deeper understanding of enzymes. Life is a sequence of ordered, tightly regulated and synchronised enzyme catalysed reactions; understanding how these reactions are carried out is a basic biological question. The success of the genome projects is presenting biologists with an immense number of novel proteins with unknown functions; we really need to know how a protein sequence leads to a structure and a structure to a function. Our interest in enzyme action is not limited to fundamental biological questions; a better understanding of enzymology has immediate technological and industrial benefits. Many drugs targets are enzyme inhibitors, but developing new drugs is a frustrating and difficult business. A better understanding of enzyme action will make it easier to discover potential drugs. In a general industrial setting the ability of enzymes to accelerate chemical reactions at low temperatures and in aqueous solution has attracted attention as designed enzymes may be able to replace expensive high temperature processes in environmentally unfriendly solvents with a cheap, low temperature, biodegradable enzyme system. My proposal is firmly directed at fundamental questions in chemical biology. Wider benefits will arise not just from the work in my group, but also from training future enzymologists in the techniques needed to address questions of enzyme action and from application to broader industrial and medical problems.

A combination of structural biology, spectroscopy, mutagenesis and kinetics will be used to reveal the role of enzyme structure in distorting and deprotonating substrate and thus accelerating the catalysed reaction. The choice of ferrochelatase as a model system allows the use of Raman spectroscopy to explore substrate distortion and transient kinetics to reveal the mechanistic consequences. We aim to (i) kinetically characterise individual events in iron chelation catalysed by a series of single and double mutant enzymes, (ii) characterize the role of active site structural contacts in distorting the bound porphyrin substrate, (iii) explore the role of active site contacts in active site substrate deprotonation and structural constraints in the putative proton shuttles and (iv) reveal the significance of residues remote from the active site in promoting transition state stabilization. We will use site-directed-mutagenesis to generate a series of structural variants of ferrochelatase predicted to vary in their ability to distort or deprotonate bound substrate. Raman spectroscopy will be used to probe substrate and product distortion. Structural constraints in active-site deprotonation will be explored using pH dependant kinetics and spectroscopy. Our newly developed transient kinetic methods will allow direct assessment of the relationship between substrate reactivity in the active site and these physiochemical parameters.