1 INTRODUCTION Enzymes are effectors of chemical change. Through their action, enzymes bring about the transformation of one chemical substance into another. Like all change, the chemical change brought about by enzymes involves a temporal aspect that can be expressed as the rate at which the change occurs. The systematic study of these rates defi nes the biochemical fi eld known as enzyme kinetics. In this book, I describe the various approaches that are used to study the kinetics of enzyme catalysis and inhibition. The chapters of this book are arranged in order of increasing complexity of the system under study, moving from single substrate enzy- matic reactions and their inhibition, to two substrate reactions and their inhibition. Along the way are chapters devoted to special areas, such as the utility and construction of free energy diagrams, kinetics of multi - intermediate reactions, and the kinetic analy- sis of tight - bindingCOPYRIGHTED and time - dependent inhibitors. MATERIALWhile this book should be useful to any investigator involved in kinetic examinations of enzymatic reactions, it is aimed at those involved in drug discovery research, where kinetic characterization is fundamen- tal to drug discovery programs that have enzymes as their therapeutic target. In this introductory chapter, I discuss three topics that set the stage for the remain- der of the book. I fi rst provide a historical context for our subsequent discussions of Kinetics of Enzyme Action: Essential Principles for Drug Hunters, First Edition. Ross L. Stein. © 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc. 1 cc01.indd01.indd 1 55/5/2011/5/2011 22:49:25:49:25 PPMM 2 INTRODUCTION enzyme kinetics. Here we will discuss the historical connections between enzymology and other branches of chemistry, early developments in the kinetics of enzymatic reac- tions, and several aspects of contemporary enzymology. In the second section of this chapter, we dissect the concept of “ mechanism of action ” into component parts, or “ sub - mechanisms, ” and see what is actually involved in elucidating an enzyme ’ s mech- anism. Finally, in the third section, we discuss how an accurate description of enzymatic mechanisms can emerge from kinetic data. 1.1 A BRIEF HISTORY OF ENZYMOLOGY The history I present below is incomplete; space does not permit a full historical account of enzymology. Rather, this summary was written to give the reader some sense of how enzymology developed into the sophisticated, quantitative science it has now become. More often than not, I let the many pioneering fi gures of enzymology speak for them- selves in quotations drawn from their original works. 1.1.1 A History of the Interplay between Organic and Biochemistry Since its beginning in the closing decades of the nineteenth century, enzymology has developed in close relation with both biochemistry and organic chemistry. Advances in organic chemistry have informed and enriched both biochemistry and enzymology, and there have always been strong synergistic interactions between enzymology and bio- chemistry. Organic and biochemistry are, of course, subdivisions that grew out of chemistry itself. Chemistry is the most ancient of the special sciences, with origins that can be traced to the dye and perfume makers of Mesopotamia, Egypt, India, and China of the third millennia B.C., and before that to metallurgists of prehistory. Following them were alchemists, who, in their search for the Philosopher ’ s Stone, fl ourished from the begin- ning of the common era on into medieval times. It was not until Robert Boyle published his The Sceptical Chymist in 1661 that the distinction between alchemy and chemistry was clearly articulated, the latter relying on the “ scientifi c method ” and inductive logic as laid out by Francis Bacon in the late sixteenth century. However, the birth of chem- istry is usually dated to Antoine Lavoisier ’ s discovery in 1783 of the law of conserva- tion of mass that refuted and laid to rest the phlogiston theory of combustion. Our chief concern in this section is with two subdivisions of chemistry, the allied fi elds of organic and biochemistry. The origin of organic chemistry is usually said to be Friedrich W ö hler ’ s synthesis of urea in 1828. This simple laboratory procedure produced the principal organic component of urine from the inorganic salt ammonium cyanate, and in one stroke dispelled the vitalistic notion that only living organisms have the ability to produce organic substances. Fully understanding the signifi cance of his accomplishment, W ö hler exclaimed to his mentor, the Swedish chemist J ö ns Jakob Berzelius: “ I can no longer, so to speak, hold my chemical water and must tell you that I can make urea without needing a kidney, whether of man or dog. ” cc01.indd01.indd 2 55/5/2011/5/2011 22:49:25:49:25 PPMM A BRIEF HISTORY OF ENZYMOLOGY 3 W ö hler ’ s landmark 1828 synthesis not only triggered a wave of interest in organic chemistry, especially in Germany, but also motivated scientists throughout Europe to study the chemical basis of biological phenomenon. This new fi eld of study would eventually become known as “ biochemistry, ” a term that would not exist until 1903, when it was coined by German chemist Carl Neuber. The true beginning of biochem- istry can be traced to the 1833 studies of French chemist Anselme Payen that resulted in the production of barley extracts that contained heat - labile components with the remarkable ability to convert starch into sugar. Previous to Payen ’ s studies, it was thought that activities such as these could occur only in intact organisms, such as the grain berry itself. The extracts that Payen investigated were called “ diastase, ” which we know now to be a mixture of related amylase enzymes. These early studies marked the beginnings of organic chemistry and biochemistry and were characterized by investigations of the macroscopic. During the infancy of biochemistry, we hear of “ substances ” and “ factors ” with no mention yet of molecules. For example, in the passage below from a publication that appeared in 1827, British physician and chemist William Prout fi rst introduces and then goes on to describe results of his studies that were aimed at understanding the “ organized bodies ” (i.e., starch, fat, and protein droplets and globules) that initially form during digestion of food stuffs and serve as the “ principal alimentary matters ” used by animals to extract nourishment. The subject of digestion had for a long time occupied my particular attention: and by degrees I had come to the conclusion, that the principal alimentary matters employed by man, and the more perfect animals, might be reduced to three great classes, namely, the saccharine [starch], the oily [fat], and the albuminous [protein]: hence, it was determined to investigate these in the fi rst place, and their exact composition being ascertained, to inquire afterwards into the changes induced in them by the action of the stomach and other organs during the subsequent processes of assimilation. It was known from the very infancy of chemistry, that all organized bodies, besides the elements of which they are essentially composed, contain minute quantities of different foreign bodies, such as the earthy and alkaline salts, iron, etc. These have usually been considered as mere mechanical mixtures accidentally present; but I can by no means subscribe to this opinion. Indeed, much attention to this subject for many years past has satisfi ed me that they perform the most important functions; in short, that organiza- tion cannot take place without them. Thus, starch I consider as merorganized sugar, the two substances having the same essential composition, but the starch differing from sugar by containing minute portions of other matters, which we may presume, prevent its constituent particles from arranging themselves into the crystalline form, and thus cause it to assume different sensible properties. (Prout 1827 ; italics in the original) We see here not a hint of Prout using molecular theory to describe the macromol- ecules (i.e., carbohydrates, lipids, and proteins) that concern him. Similarly, German chemist Moritz Traube explained fermentation and related pro- cesses in terms of substances and not molecules: “ The putrefaction and decay ferments are defi nite chemical compounds arising from the reaction of the protein substances with water, arising thus from a chemical process ” (Traube 1858a ). We see here that cc01.indd01.indd 3 55/5/2011/5/2011 22:49:25:49:25 PPMM 4 INTRODUCTION while Traub recognized that the underlying bases of fermentation and putrefaction were chemical, he did not describe the results of his experiments in molecular terms as the chemical transformation of molecules. Like Traub, many chemists of the mid - nineteenth century were reluctant to accept the existence of entities that that they could not see with their own eyes. This reluctance was despite the fact that Amedeo Avogadro fi rst proposed the existence of molecules in 1811 and the enormous inroads that Friedrich Kekul é made from 1850 – 1870 into the understanding of carbon ’ s multivalency and thus its ability to form complex struc- ture; that is, “ molecules. ” It would not be until the beginning of the twentieth century that molecular theory, fi nally embraced by most organic chemists during the closing decades of the nineteenth century, would become part of the interpretational apparatus of biochemical studies. In 1902, Franz Hofmeister reports that “ the protein molecule is mainly built - up from amino acids ” (Hofmeister 1902 ; italics mine). Three years later, a paper appeared in fi rst volume of the American publication Journal of Biological Chemistry by biochemist Phoebus A. T. Levene that extended Hofmeister ’ s observations to try to understand how differences in amino acid composition of various proteins render them more or less susceptible to degradation by the action of trypsin and other digestive enzymes of the gut.