ENZYMES AS REAGENTS 



far removed from true cellular reality. But the enzymologist 

 has learned a great deal about the mechanism of enzyme action 

 from studies with coenzymes and enzymes as reactants. 



METHODS OF ANALYSIS OF ENZYMES AS REACTANTS 



In most instances, enzyme-substrate compounds appear to be 

 unstable. The first direct demonstrations of their existence de- 

 pended on the use of rapid spectrophotometric methods. The 

 appearance of new absorption bands in the case of peroxidases 

 (4) and the disappearance of the DPN-enzyme absorption band 

 of glyceraldehyde-3-phosphate dehydrogenase (38) on addition 

 of the respective substrates, are examples of such studies. On 

 the other hand, the few enzyme-coenzyme compounds investi- 

 gated so far appear to be quite stable (38,49,50). Inhibitors 

 such as /?-chloromercuribenzoate, iodoacetate, and hydroxyl- 

 amine have been most useful for the analysis of enzyme-substrate 

 and enzyme-coenzyme interactions (16,38,49). Isotopically 

 labeled substrates have also been valuable tools in these studies. 

 With C^'*-labeled acetyl phosphate, radioactive acyl glyceralde- 

 hyde-3-phosphate dehydrogenase was prepared. Hydrolysis 

 with proteolytic enzymes resulted in the release of a thiol ester 

 with the properties of acetyl glutathione (24). Formation of a 

 phospho-enzyme in the case of phosphoglucomutase was first 

 suggested by studies with P^^ (14). The use of isotopically 

 labeled substrates or cofactors in incomplete systems (omitting 

 either a substrate or a cofactor), which frequently results in an 

 enzyme-catalyzed redistribution of the label, helps to elucidate 

 reaction sequences. Emphasis should be placed, however, on 

 the use of pure systems for such studies so that exchange reac- 

 tions, due to either contaminating coenzymes or contaminating 

 enzymes, can be ruled out. Important conclusions regarding 

 the mode of enzyme action have been deduced from studying the 

 point of cleavage with isotopically labeled substrates {cf. 21). 



Finally, valuable information has been obtained from kinetic 

 investigations of the complexes formed between enzymes, co- 

 enzymes, and substrates. It has made feasible a bold study of 



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