Kinetics of Enzyme Action

Essential Principles for Drug Hunters

Ross L. Stein

• Publisher: Wiley
• Year: 2011
• Print ISBN: 9780470414118
• eBook ISBN: 9781118084403
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Molecular Biology
• Index: Biological Sciences

Few scientists have the knowledge to perform the studies that are necessary to discover and characterize enzyme inhibitors, despite the vested interest the pharmaceutical industry has in this field. Beginning with the most basic principles pertaining to simple, one-substrate enzyme reactions and their inhibitors, and progressing to a thorough treatment of two-substrate enzymes, Kinetics of Enzyme Action: Essential Principles for Drug Hunters provides biochemists, medicinal chemists, and pharmaceutical scientists with numerous case study examples to outline the tools and techniques necessary to perform, understand, and interpret detailed kinetic studies for drug discovery.

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 defines the biochemical field 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 enzymatic 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 analysis of tight-binding and time-dependent inhibitors. While 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 fundamental 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 remainder of the book. I first provide a historical context for our subsequent discussions of enzyme kinetics. Here we will discuss the historical connections between enzymology and other branches of chemistry, early developments in the kinetics of enzymatic reactions, 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 mechanism. 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 figures of enzymology speak for themselves 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 biochemistry. 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, flourished from the beginning 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 “scientific method” and inductive logic as laid out by Francis Bacon in the late sixteenth century. However, the birth of chemistry is usually dated to Antoine Lavoisier’s discovery in 1783 of the law of conservation 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 fields of organic and biochemistry. The origin of organic chemistry is usually said to be Friedrich Wöhler’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 significance of his accomplishment, Wöhler exclaimed to his mentor, the Swedish chemist Jöns 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.”

Wöhler’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 field 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 biochemistry 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 first 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 first 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 satisfied me that they perform the most important functions; in short, that organization 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 macromolecules (i.e., carbohydrates, lipids, and proteins) that concern him.

Similarly, German chemist Moritz Traube explained fermentation and related processes in terms of substances and not molecules: “The putrefaction and decay ferments are definite chemical compounds arising from the reaction of the protein substances with water, arising thus from a chemical process” (Traube 1858a). We see here that 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 first proposed the existence of molecules in 1811 and the enormous inroads that Friedrich Kekulé made from 1850–1870 into the understanding of carbon’s multivalency and thus its ability to form complex structure; that is, “molecules.”

It would not be until the beginning of the twentieth century that molecular theory, finally 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 first 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. Levene remarks that “polypeptides composed of the lower amino-acids are decomposed by trypsin less readily than polypeptides containing in their molecule the higher acids” (Levene 1905; italics mine).

Organic chemistry and biochemistry were rapidly becoming molecular sciences. During the twentieth century, organic chemistry would develop in many directions, spawning a host of subspecialties (e.g., synthetic organic chemistry, physical organic chemistry, organo-metallic chemistry) and entire industries (e.g., polymers, pharmaceuticals, petroleum products). At the same time, biochemists would unravel the intricacies of the many metabolic pathways that comprise cellular physiology, and probe the structure and function of the principal macromolecules of all living organisms—DNA, RNA, proteins, carbohydrates, and lipids. The history of the development of organic chemistry and biochemistry in the twentieth century is a fascinating story, in which the two disciplines at times separate to only merge again several years later in their intertwined and symbiotic relationship.

1.1.2 Early Developments in the Quantitative Study Enzyme-Catalyzed Reactions: Kinetics, Catalysis, and Inhibition

Even in its infancy, enzymology possessed a quantitative aspect that organic chemistry and biochemistry largely lacked. A key concept that allowed the development of enzymology as a quantitative science was the idea that enzymes are chemical in nature, “definite chemical compounds” (Traube 1858b). Wilhelm Kuhne, who coined the term enzyme, explained that enzymatic reactions are “simple chemical changes” and that enzyme “activity can occur without the presence of the organisms and outside the latter” (Kuhne 1877). Buchner, in his studies of fermentation, insisted that “an apparatus as complicated as the yeast cell is not required to institute the fermenting process” (Buchner 1897). He also had the insight that “the carrier of the fermenting activity of the press juice must be a dissolved substance, undoubtedly a protein.” (Buchner 1897).

It is interesting to note that even in the face of these advances toward establishing the chemical nature of enzymes, there persisted the sense that enzymes must still possess, in some manner, the “vital force” of the organism from which they were extracted. For example, in 1901, Joseph Kastle published a paper in Science entitled “On the Vital Activity of the Enzymes,” where he concluded that “the enzymes are active in the same sense of retaining certain of the vital activities of the living cell” (Kastle 1901).

1.1.2.1 Chemical Kinetics, the Concept of the Active Site, and Enzyme Kinetics.

One of the goals of enzymology, both then and now, is to establish quantitative and predictive relationships between reaction velocities and experimental variables, such as enzyme and substrate concentration. Methods to accurately measure the rates of enzymatic reactions and the sense that these measurements could be made reproducibly and lead to testable hypotheses concerning how reaction rates depend on experimental variables, grew out of the rapidly evolving field of chemical kinetics.

Chemical kinetics was born with Ludwig Wilhelmy’s 1850 publication on the kinetics on the acid-catalyzed hydrolysis of sucrose (Wil...

Table of contents

• Cover
• Title page
• Copyright page
• DEDICATION
• PREFACE
• 1 INTRODUCTION
• 2 KINETICS OF SINGLE-SUBSTRATE ENZYMATIC REACTIONS
• 3 KINETICS OF SINGLE-SUBSTRATE ENZYMATIC REACTIONS: SPECIAL TOPICS
• 4 ENZYME INHIBITION: THE PHENOMENON AND MECHANISM-INDEPENDENT ANALYSIS
• 5 KINETIC MECHANISM OF INHIBITION OF ONE-SUBSTRATE ENZYMATIC REACTIONS
• 6 TIGHT-BINDING, SLOW-BINDING, AND IRREVERSIBLE INHIBITION
• 7 KINETICS OF TWO-SUBSTRATE ENZYMATIC REACTIONS
• 8 KINETIC MECHANISM OF INHIBITION OF TWO-SUBSTRATE ENZYMATIC REACTIONS
• 9 ALLOSTERIC MODULATION OF ENZYME ACTIVITY
• 10 KINETICS-BASED PROBES OF MECHANISM
• APPENDIX A: BASIC PRINCIPLES OF CHEMICAL KINETICS
• APPENDIX B: TRANSITION STATE THEORY AND ENZYMOLOGY: ENZYME CATALYTIC POWER AND INHIBITOR DESIGN
• APPENDIX C: SELECTING SUBSTRATE CONCENTRATIONS FOR HIGH-THROUGHPUT SCREENS
• Index