The Effect of Divalent Metal Ions on the Rate and Transition-State Structure of Phosphoryl-Transfer Reactions’

The divalent metal ions, Mg2+ and Ca”, catalyze the reaction of p-nitrophenyl phosphate dianion (PNPP) with substituted pyridines but do not increase the associative character of the transition state, as indicated by values of p,,, in the range 0.17-0.21 for PNPP and for its metal ion complexes. The reactions of phosphorylated morpholinopyridine (MPP) are inhibited approximately twofold by Mg2+ and Ca2+. This shows that there is no electrophilic catalysis from the interaction of a metal ion with the phosphoryl oxygen atoms and is consistent with electron donation from the phosphoryl oxygen atoms in a dissociative transition state. The constant value of PI, = -1.02 to -1.05 for the hydrolysis of three phosphorylated pyridines in the presence and absence of 0.33 M Mg2+ and the constant ratio of the rate constants for the reactions of two pyridines with MPP and MPP-Mg also provide no indication of a significant change in transition-state structure upon binding of the metal ion. The dependence of the observed rate constants on the concentration of the metal ions and substrates at ionic strength 1.0 indicates the formation of 1:l complexes with K,(PNPP.Mg) = 14.8 M-’ and K,(PNPPCa) = 7.5 M-I at 39.2 OC and K,(MPP.Mg) = 4.4 and 9.1 M-’ at 25.1 and 52.3 OC, respectively. The threeto sixfold catalysis by Mg2+ and Ca2+ of the reaction of PNPP with pyridines contrasted, with the inhibition of reactions with uncharged leaving groups, suggests that the bound metal ion interacts with the phenoxide ion leaving group as well as the phosphoryl oxygen atoms in the transition state. 2,4-Dinitrophenyl phosphate dianion (DNPP) binds Ca2+ and Mg2+ with kinetically determined association constants of 6.0 and 6.2 M-’, respectively, at 39.1 O C and ionic strength 1.0, and DNPP-Ca reacts with pyridines sevenfold faster than free DNPP. The constant value of p,,, for the reactions of DNPP and DNPPCa with two substituted pyridines again gives no evidence for a significant change in nucleophilic participation upon complexation with a divalent metal ion. The reactions of DNPPSMg with pyridines are at least eightfold slower than the reactions of free DNPP because of an unfavorable interaction of Mg2+ and the ortho nitro group of DNPP in the transition state. The effects of Mg2+ and Ca2+ on the rate constants for hydrolysis follow the same trends as for the reactions with pyridines, except that the hydrolysis of PNPPeMg is 20% slower than that of PNPP. The mechanisms employed by enzymes to catalyze phosphoryl-transfer reactions are not understood. It has often been suggested that complexation with a metal ion could change the mechanism of phosphoryl transfer from the largely dissociative mechanism observed in the absence of a metal ion to an associative mechanism, with more nucleophilic involvement in the transition state.” A change from a dissociative to an associative mechanism (1) Supported in part by grants from the National Institutes of Health (GM20888 and AM07251) and the National Science Foundation (PCM8117816). D.H. was supported by a Fellowship of the Gillette Foundation. (2) See, for example: Mildvan, A. S.; Fry, D. C. Adu. Enzymol. 1987,59, 241-313. Mildvan, A. S . Adu. Enzymol. 1979, 49, 103-126. Mildvan, A. S.; Grisham, C. M. Struct. Bond. 1974, 20, 1-21. Benkovic, S. J.; Schray, K. J. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York; 1973; Vol. 8, pp 201-238. Williams, A,; Naylor, R. A. J . Chem. SOC. B 1971, 1913-1979. 0002-7863/87/1509-4665$01.50/0 would allow a larger potential advantage from catalysis by induced intramolecularity in an enzymatic reaction because reactions with “tight” transition states have stricter requirements for alignment than reactions with “loose” transition ~ t a t e s . ~ In addition, a change to an associative mechanism would give a greater potential advantage from general base catalysis due to the increased bond formation to the nucleophile in the transition state and concomitant charge development on the nucleophile. W e have used a nonenzymatic system to test whether or not complexation with a metal (3) Benkovic, S. J.; Dunikoski, L. K., Jr. J . Am. Chem. SOC. 1971, 93, (4) Kirby, A. J.; Jencks, W. P. J . Am. Chem. SOC. 1965,87, 3209-3216. (5) Kirby, A. J. Adu. Phys. Org. Chem. 1980, 17, 183-278. Page, M. I . Angew. Chem., Inr. Ed. Engl. 1977, Id, 449-459. Page, M. I. Chem. SOC. Reu. 1973, 2, 295-323. Jencks, W. P. Adu. Enzymol. 1975, 43, 219-410. 1526-1527.