ENZYMES AS REAGENTS 



esteratic sites, according" to their interactions witli the quaternary 

 ammonium and the ester moiety of the substrate. A theory of a 

 two-step mechanism of action was proposed which included 

 the rate-Hmiting formation of an acyl enzyme as an intermediate. 

 The acyl group can be hydrolyzed or transferred to an acceptor 

 other than water, e.g., hydroxylamine or alcohol. The in- 

 activation of acetylcholinesterase by inhibitory phosphate esters, 

 such as tetra alkyl pyrophosphate, was shown to be an enzymatic 

 process reversible by suitable nucleophilic replacing agents. 



SUBSTRATE-SUBSTR.\TE INTERACTIONS 



Theories of the mode of action of glyceraldehyde-3-phos- 

 phate dehydrogenase and glutamic dehydrogenase which assume 

 interaction between substrates prior to enzyme action have been 

 widely accepted, although no experimental evidence for chemical 

 addition products as intermediates is available. In the case of 

 glyceraldehyde-3-phosphate, it was assumed that phosphate 

 adds to the aldehyde prior to its oxidation to an acyl phosphate 

 {cf. 52). A useful kinetic approach to this problem has been 

 made by Biicher and Garbade (3). The experimental data on 

 the effect of phosphate or arsenate on the apparent Kr„ value for 

 the substrate (glyceraldehyde or glyceraldehyde-3-phosphate) 

 seem to rule out the formation of a glyceraldehyde diphosphate 

 as intermediate. Strecker has presented (45) evidence of a 

 similar nature against the chemical formation of the a-imino 

 compound as intermediate in the reductive amination of a-keto- 

 glutarate to glutamate. 



Enzymes as Analytical Tools 



Analysis of biochemical events is dependent on the avail- 

 ability of analytical tools. Isolation procedures and chemical 

 determinations often lack accuracy and specificity. Enzymolo- 

 gists have naturally turned to enzymes as an aid in assays of 

 intermediary metabolites. Kjeldahl, over 70 years ago (19), 

 was probably the first to propose the use of an enzyme as an 

 analytical tool (invertase for sucrose deterixiination). Since 

 then, numerous enzymes have been used for the assay of sub- 

 strates, coenzymes, and enzymes. The obstacle of an unfavor- 



225 



