Active-site-directed reagents

Having an increased or elevated reactivity. This term has been used in reference to the relative activity of amino acyl residues at the active sites of enzyme. The immediate environment (Le., the microenvironment) may allow simple reagents to react faster with the amino acid than would normally be expected. Thus, in labeling of proteins with active site-directed reagents, an investigator should always consider the basis of increased reactivity Is it due to facilitation of the reaction by increased affinity (Le., affinity labeling), or is it due to increased activity of the amino acyl side chain (e.g., perhaps increased nucleophilicity due to the microenvironment). 

A mechanism seen on occasion in affinity labefing experiments when an active site-directed reagent binds to the active site of an enzyme and protects that active site against a bimolecular reaction with a second mofecufe... 

Chemical Modification by Active-Site-Directed Reagents Elliott Shaw... 

E. Shaw inP. D. Boyer, Ed, Chemical Modification by Active-Site Directed Reagents, Acch demic Press, New York, 191Q,pp. 91-147. 

Relatively little is known about the physical properties of ACAT. Enzyme activity has been solubilized from different tissues and reconstituted into lipid vesicles, but has been purified only partially [24,25]. Even the kinetics and mechanism of the ACAT reaction are poorly understood. As in the case of most enzymes involved in lipid metabolism, kinetic constants are difficult to determine because the substrates are water insoluble and membrane associated [26]. Moreover, systematic efforts to identify the active site of the enzyme through the use of active site-directed reagents have yet to be made. Such efforts might be worthwhile even at this stage of investigation of the enzyme because the use of radioactive, site-directed reagents might allow identification of the enzyme protein. 

Studies on the inactivation mechanism of MAO by active site-directed reagents are potentially useful in defining the chemical mechanism of the normal reaction. In this review, only compounds known to be mechanism-based inhibitors will be studied, particularly in their relationship to elucidating the chemical mechanism of this enzymic redox reaction. 

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