Kinetic Analysis of Macromolecules: A Practical Approach

Rigorous kinetic analysis combined with high-resolution structural data can allow the direct assessment of the functions of proteins and nucleic acids, but transient kinetic experiments must be adapted to the system under study, and it is often difficult to know in advance what methods to use to measure single reaction cycles midst the noise of the ongoing cascade of reactions. Chemists, most of them biochemists, describe a variety of systems for doing so. Among these are using rapid chemical quench methodology and single enzyme turnover experiments to detect and characterize enzyme intermediates, a fluorescent sensor to assay inorganic phosphate, using competition experiments to determine binding parameters, and time-resolved spectral analysis. Annotation ©2004 Book News, Inc., Portland, OR

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Reviewer:Eugene A Davidson, PhD(Georgetown University School of Medicine)
Description:This is a collection of articles/protocols covering detailed kinetic analysis of macromolecular interactions. The main thrust is to provide technical directions for kinetic analysis of enzymatic reactions.
Purpose:This book introduces the reader to kinetic analyses beyond the customary steady state measurements usually employed. In so doing, the opportunity to define mechanisms as well as rate determining steps is made clear. In terms of a more thorough analysis of enzyme reactions, this is worthwhile. Although each of the articles uses a specific reaction type for analysis, general principles can nonetheless be learned. In that sense, the goal of the book is achieved.
Audience:It is intended for the enzyme mechanist or drug developer with a strong background in conventional kinetics. Graduate students working in this area, postdoctoral fellows and laboratory directors will find material of value. The editor has assembled a qualified set of authors.
Features:Since the introduction of Michaelis-Menten kinetics (and subsequent refinements) beginning about 90 years ago, most kinetic analysis of enzymatic reactions has been dominated by that approach. Few investigators go beyond determination of the classic constants, Km and kcat. More recently, those studying details of enzyme mechanisms have employed isotopes such as deuterium or oxygen-18 to identify rate-determining steps or the origin of a specific oxygen. This book extends such analyses to the realm of transient kinetics wherein analysis of early stages of reactions (usually in the millisecond range) can provide information about possible intermediates (or rule out some) and therefore valuable information about the reaction path. Approaching the problem from a kinetic perspective, the opening chapter provides a valuable overview of the reasons for this type of approach. The remaining chapters offer protocols for various types of enzymatic reactions. Included is a chapter on the use of a modified enzyme as an analytical sensor (for inorganic phosphate) and one on enzymes that have specific residues replaced. Although each of the associated protocols deals with the specific system under study (ribozyme cleavage, DNA-protein interactions etc.), general principles are clearly there. Application to aspects of drug development is clearly delineated in the section on quench methodology (focus is on reactions of phosphoenolpyruvate). The investigator wishing to identify possible intermediates in a reaction pathway will find much useful information. This book can also be recommended to students since it will introduce them to the next level of analysis -- something of value for the next generation of enzymologists. Regrettably, the title is somewhat obscure and could be better edited to read Kinetic Analysis of Macromolecular Interactions.
Assessment:In spite of a somewhat distracting title, this is a useful book. The technical requirements for the work described may, on occasion, be formidable, but the information gained cannot be easily acquired otherwise. A useful contribution.