ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 266, No. 1, October, pp. 132-141, 1988 Pyrophosphate Formation from Acetyl Phosphate and Orthophosphate: Evidence for Heterogeneous Catalysis’ JOSE ROBERTO MEYER-FERNANDES* AND ADALBERTO VIEYRAyZ *Departamento de Biologia C&h e Tecidual, Universidade do E&ado do Rio de Janeiro, 20550 Rio de Janeiro, Brazil; and TDepatiamento de Bioquimica, Institute de Ci&a.q Biom&icas, Centro de Ci&ci~ da Sazide, Universidade Federal do Rio de Janeiro, RJ, 21910 Rio de Janeiro, Brazil Received February 9,1988, and in revised form June 1,198s The formation of [32P]pyrophosphate from acetyl phosphate and [32P]orthophosphate was studied under conditions in which phosphate-metal salts or acetyl phosphate-metal salts precipitate. In the absence of precipitates in purely aqueous media, the initial rate constant of transphosphorylation (/cobs) was extremely small and the formation of pyro- phosphate was detected only in the presence of calcium. In various combinations, condi- tions such as high pH, high concentrations of reactants, and the presence of dimethyl sulfoxide caused three types of precipitates to form. In completely aqueous solution with an excess of orthophosphate, the crystals formed at high pH contained 3 mol of calcium for 2 mol of phosphate and they were poorly effective at promoting phosphorolysis. In the presence of dimethyl sulfoxide, the ratio of calcium to phosphate in the sediment was 1:l and phosphorolysis proceeded at a high rate. In either solvent, an excess of acetyl phosphate caused precipitation of a complex containing 1 mol of acetyl phosphate to 1 mol of calcium. In aqueous media the rate constant of phosphorolysis increased with increasing precipitation of the acetyl phosphate-calcium complex. With destabilization of the anions by dimethyl sulfoxide the increase in lcobs for a given amount of acetyl phosphate-calcium precipitated was 200-fold higher. Magnesium did not form precipi- tates and was ineffective in promoting transphosphorylation in completely aqueous me- dia, either in the presence of excess phosphate or in the presence of excess acetyl phos- phate. However, when precipitation of phosphate-magnesium or acetyl phosphate-mag- nesium was promoted by addition of dimethyl sulfoxide, phosphorolysis was observed with rate constants as high as those found in the presence of calcium. These results indicate that phosphorolysis of acetyl phosphate occurs at higher rates on the surface of solid structures, through highly specific interactions involving acetyl phosphate, or- thophosphate, and divalent cations. 019aAc~d~mic~re~~,1~~. The formation of PPi3 in the absence of added divalent cations in media containing concentrated AcP was reported many years ago (1). Recently (2), we demon- ’ Supported by grants from Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq), Fi- nanciadora de Estudos e Projetos (FINEP), and Con- selho de Ensino para Graduados (CEPG/UFRJ), Brazil. ’ To whom correspondence should be addressed. 3 Abbreviations used: PPi, pyrophosphate; AcP, acetyl phosphate; PEI-cellulose, poly(ethyleneimine)- strated that “2PPi formation in the pres- ence of several divalent cations depends on the concentration of both reactants AcP and 32Pi, and postulated that it could pro- ceed through a nucleophilic attack of Pi on the acyl phosphate moiety of the AcP mol- ecule. In this reaction, the chemical energy of the acyl phosphate bond is conserved in the phosphoanhydride linkage of pyro- cellulose; Pi, orthophosphate; MeaSO, dimethyl sulf- oxide; Tris, tris(hydroxymethyl)aminomethane. 0003-9861/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form resewed. 132