Affinity labels, design

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Since the discovery that glycolate was an alternate substrate for pyruvate kinase ( ), several other o-hydroxy acids have also been found to be substrates for this enzyme ( ). This class of alternate substrates provides a new approach the problem of substrate specificity for pyruvate kinase. 3-Nitrolactate is one such alternate substrate. Interestingly, the phosphorylated product of this reaction inactivates the enzyme (86). However, 3-nitrolac-tate does not behave as a straightforward affinity label since covalent modification occurs nonspecifically. It is hoped that this new Information may lead to the design of an affinity label of this enzyme, further serving to pinpoint amino acid groups at the active site. 

In the course of studying the mechanism of action of creatine kinase from rabbit skeletal muscle (M.M isoenzyme), Kenyon and coworkers (4,90) have been involved in the design of specific irreversible inhibitors that are active-site-directed (affinity labels). Creatine kinase catalyzes the reversible transfer of a phosphoryl group ( the elements of "POi") from ATP to creatine, as shown in the following reaction ... 

Designing specific enzyme inhibitors on a rational basis when one does not have a detailed three-dimensional crystal structure to which to relate is a rather sophisticated challenge. Some viable approaches to such a challenge are discussed in a review chapter by Santi and Kenyon (91) This discussion will focus on our rationale for the design of an affinity label for creatine kinase, namely N-(2,3-epoxypropyl)-N-amidinoglycine (epoxycreatine ) ... 

An affinity label, or active-site-directed irreversible inhibitor, is a chemically reactive compound that is designed to resemble a substrate of an enzyme, so that it binds specifically to the active site and forms covalent bonds with the protein residues.1-3 Affinity labels are very useful for identifying catalytically important residues and determining their pKa values from the pH dependence of the rate of modification. 

CONTENTS Introduction to the Series An Editor s Foreword, Albert Padwa. Preface, Bruce E. Maryanoff and Cynthia A. Maryanoff. Computer Assisted Molecular Design Related to the Protein Kinase C Receptor, Paul A. Wenderand Cynthia M. Cribbs. Chemistry and Biology of the Immunosuppressant (-)-FK-506, Ichiro Shinkai and Nolan H. Sigal. The Development of Ketorolac Impact on Pyrrole Chemistry and on Pain Therapy, Joseph M. Muchowski. Application of Silicon Chemistry in the Corticosteroid Field, Douglas A. Livingston. Hu-perzine A-A Possible Lead Structure in the Treatment of Alzheimers Disease, Alan P. Kozikowski, X.C, Tang and Israel Hanin. Mechanism-Based-Dual-Action Cephalosporins, Harry A. Albrecht and James G. Christenson. Some Thoughts on Enzyme Inhibitors and the Quiescent Affinity Label Concept, Mien Krantz Index. 

Chemical modification can be used to obtain information on the catalytic mechanism and on the catalytic site of the enzyme of interest. One goal in the design of affinity labels for enzymes is to determine the catalytically important residues. First, the affinity label has to behave as an analogue of the substrate (or of the activator or inhibitor) by competition experiments. 

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