Stereochemistry of Phospho and Nucleotidyl Transfer

Stereochemical information is important in the analysis of most reaction mechanisms. This is true for all substitution reactions at atoms with substituents in tetrahedral array, such as saturated carbon and tetravalent phosphorus. Enzymic substitution at phosphorus in phosphoric esters and phosphoanhydrides is not an exception to the rule. There are experimental complications, however, in that all naturally occurring biological phosphates have two or more chemically equivalent oxygens, so that none has chirally substituted phosphorus. Inasmuch as an asymmetric arrangement of substituents is required for stereochemical analysis, P-chiral substrates for stereochemical studies of phosphotransferases and nucleotidyltransferases must be synthesized with sulfur or heavy isotopes of oxygen as substituents in an asymmetric array. 

The methodologies for synthesis and configurational analysis of P-chiral biological phosphates are extensively reviewed elsewhere and will not be repeated here (2-7). Most and perhaps all biological phosphates can be synthesized with a chiral phosphorus, and the configuration about chiral P in any enzymic product can probably be determined by spectroscopic and chromatographic methods. 

Two kinds of information about nucleotidyltransferases and phosphotransferases are obtained by use of substrates or substrate analogs with chiral P. The stereochemical course of phosphoryl transfer and nucleotidyl transfer gives important information about the reaction mechanism. If inversion of configuration at phosphorus is observed, it may be concluded that an uneven number of displacements at phosphorus occurs in the reaction mechanism. If retention of configuration at phosphorus is observed, it may be concluded that the mechanism entails an even number of displacements at phosphorus. Inversion corresponds to the single-displacement mechanism of Eq. (2), and retention indicates a mechanism such as that of Eq. (3) or Eqs. (4a) and (4b). 

A few specific cases of stereochemical analysis exemplify the use of stereochemistry to analyze phospho transfer mechanisms. UDPglucose pyrophosphor-ylase catalyzes the interconversion of (/ p)-UTPaS and (5p)-UDPaS-glucose, as shown in reaction (5) (9, 10). 

The configuration about P is inverted in this reaction, which must, therefore, proceed by a mechanism involving an uneven number of displacements at P, most likely one in this case. Adenylate kinase catalyzes reaction (6), a [ 0] thiophospho transfer from (/ p)-[7- 02]ATPyS to AMP to form (5p)-[/3- 0] ADP S (11). 

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