Interaction of phosphate analogs with glyceraldehyde-3-phosphate dehydrogenase

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Abstract
The glycolytic enzyme [yeast] glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate. A variety of phosphonates substitute for phosphate in this reaction. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Bronsted coefficient, .beta.eq, of .apprx. 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molbydate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The 2nd-order rate constant for the reaction with molybdate is only .apprx. 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25.degree. C), the half-life for hydrolysis of acyl molybdate, or the acyl arsenate, is less than 2.5 s. Hybrolysis of 1-molybdo- and 1-arseno -3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same condtions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO42- and SeO42-). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. At least 1 anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme are apparently essential for catalysis.