Phosphoryl transfer kinetic analysis

A recent kinetic study of the catalysis of hydrolysis of phosphorylated pyri-dines by Mg in aqueous solution as a model for enzymic phosphoryl transfer reactions has been reported by Herschlag and Jencks (23). They have provided evidence that a l(y -10 rate enhancement can be obtained in the presence of Mg +. From the analysis, approximately a l(y -fold rate enhancement can be attributed to the greater nucleophilicity of the species Mg(OH)+ compared to... 

The emphasis in kinetic studies of E-IIs has been on the analysis of the rates of phosphorylation of the sugar by the phosphoryl group donor. In the early studies the question was addressed whether phosphorylated E-II would be a catalytic intermediate in the reaction or whether the phosphoryl group would be transferred directly from the donor to the sugar on a ternary complex between the enzyme and its substrates [66,75,95-100]. This matter has been satisfactorily resolved by a number of other techniques in favor of the first option and possible reasons why some systems did not behave according to a ping-pong type of mechanism have been discussed [1]. 

Despite the fact that reaction kinetics cannot be Ping-Pong, the enzyme is phosphorylated by substrates and the phosphoenzyme reacts with ADP to form ATP. Is this process a part of the mechanism of reaction (16) The stereochemical analysis of phospho transfer argues strongly against the importance of the phosphoenzyme in the reaction mechanism. The phospho transfer proceeds with inversion of configuration at phosphorus, which is consistent with a single displacement at phosphorus and inconsistent with a double-displacement mechanism (64). Therefore, the phosphoenzyme does not appear to be on the main catalytic pathway. 

As is known for other nucleoside kinases, the steady-state kinetics of the adenosine kinase reaction is complex owing to regulatory effects. Adenosine and AMP apparently bind at a regulatory site, where they modulate activity, as well as at the active site, where they act as substrates. These interactions complicate the kinetics, but a careful analysis shows that the basic kinetic pathway is sequential and involves the compulsory formation of ternary complexes (79). Thus, the kinetics is consistent with the stereochemistry and suggests that the phospho transfer is a direct, one-step displacement between substrates bound at the active site in a ternary complex. Complications introduced into the mechanistic analysis of this enzyme by the adventitious phosphorylation of the protein by ATP have been discussed elsewhere (7). 

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