Patricia A. Benkovic

The binding of products, metal ion, and a substrate analog to rabbit liver fructose bisphosphatase

Abstract Binding of fructose-6-P and Pi to rabbit liver fructose bisphosphatase has been analyzed in terms of four negatively cooperative binding sites per enzyme tetramer. The association of fructose-6-P occurs in the absence of divalent metal ion, although the extent of binding is increased in the order Mg2+ d -fructofuranoside-1,6-P2 in the presence of Mn2+ closely resembles that found for fructose-1,6-P2 in the absence of Mn2+, although the measured constants are on average an order of magnitude smaller. Combination experiments with the three ligands show that the binding follows an identical ordered sequence, i.e., the tighter sites are initially occupied regardless of the ligands identity. The binding of Pi or fructose-6-P is not altered by the presence of the other. Comparison of binding constant with Ki values obtained from steady-state assays permits identification of the catalytic sites expressed in the latter. The association of Mn2+ at the catalytic site can be induced by fructose-6-P or the substrate analog suggesting that a 1-phosphoryl group enhances but is not necessary for Mn2+ binding at this site. The binding of AMP is decreased in the presence of substrate analog relative to fructose-1,6-P2, suggesting that the 2-hydroxyl serves as a “molecular signal.” From the single and combined binding experiments, a calculation of the equilibrium constant for the overall hydrolysis reaction on the enzyme surface in the presence of Mn2+ has been carried out and an estimate made for the Mg2+ case.

The synthesis and properties in enzymic reactions of substrate analogs containing the methylphosphonyl group

Abstract The properties of the methylphosphonyl group as a substrate analog for the phosphoryl moiety of various biological phosphoryl donors have been investigated in several enzymic phosphoryl transfer reactions. The synthesis and characterization of adenosine 5′-[β-methylphosphonyl]diphosphate, adenosine 5′-methylphosphonate, acetyl methylphosphonate, and methylphosphonoenolpyruvate are fully described. Adenosine 5′-[β-methylphosphonyl]diphosphate is not a substrate for adenylate kinase, hexokinase, 3-phosphoglycerate kinase, glycerol kinase, phosphofructokinase, creatine kinase, alkaline phosphatase, or nucleoside 5′-diphosphate kinase. Competitive inhibition of ATP was observed with hexokinase and 3-phosphoglycerate kinase with K i K m ∼- 10 . Adenosine 5′-methylphosphonate was a substrate for adenylate deaminase and 5′-nucleotidase, but not for adenylate kinase, acid phosphatase, 5′-phosphodiesterase, or 3′-phosphodiesterase. Acetyl methylphosphonate inhibits the reaction of acetyl phosphate with acetate kinase, but methylphosphonoenolpyruvate has no effect upon the reaction of phosphoenolpyruvate with pyruvate kinase. The results indicate that with the exception of 5′-nucleotidase, the methyphosphonyl group is incapable of undergoing phosphoryl transfer. One interpretation among others is that a metaphosphate-type mechanism is required for these processes.

Mechanism of hydrolysis of phosphorylethanolamine diesters. Intramolecular nucleophilic amine participation

Intramolecular displacement reactions at phosphorus have been examined in a series of N-alkyl-O-arylphosphorylethanolamines in water at 35 °C. The examination of the pH–rate profiles and the direct observation by 31P n.m.r. of the reaction products implicate a nucleophilic role for the amine. A rate enhancement of 106–107 is observed. Structure–reactivity correlations derived by changing the pKa of the amine and leaving group yield values for βnuc≃ 0.7 and β1g≃–1.25 and support an uncoupled concerted mechanism. A discussion of the mechanisms of nucleophilic reactions involving amines and oxyanions with inter- and intra-molecular phosphate di- and tri-esters is presented.