The substrate reactivity of mu-monothiopyrophosphate with pyrophosphate-dependent phosphofructokinase: evidence for a dissociative transition state in enzymatic phosphoryl group transfer.
mu-Monothiopyrophosphate (MTP), an analogue of pyrophosphate (PPi) with sulfur in place of oxygen in the bridge position, is a substrate for the enzyme pyrophosphate-dependent phosphofructokinase. At pH 9.4 and 6 degrees C, the maximal velocity for the phosphorylation of fructose 6-phosphate (F6P) by MgMTP is about 2.8% of that with MgPPi as the phosphoryl donor. The kinetic mechanism is equilibrium random with rate-limiting transformation of the substrate ternary complex to the product when either MgMTP or MgPPi is the phosphoryl donor. This is known from independent studies to be kinetic mechanism at pH 8.0 and 25 degrees C [Bertagnolli, B. L., & Cook, P. F. (1984) Biochemistry 23, 4101-4108]. The dissociation constant of MgPPi is 14 microM, that of MgMTP is 64 microM, and that of F6P from the enzyme is about 5 mM. The Km values for MgPPi and MgMTP are 14.5 and 173 microM, respectively. MgMTP competes with MgPPi for binding to the enzyme. The values of kcat are 3.4 s-1 and 140 s-1 for MgMTP and MgPPi, respectively, at pH 9.4 and 6 degrees C. The estimated rate enhancement factors are 3.6 x 10(5) and 1.4 x 10(14) for the reactions of MgMTP and MgPPi, respectively. Therefore, MgMTP is a reasonably good substrate for PPi-dependent PKF, on the basis of comparisons of kcat. However, the rate enhancement factors show that the enzyme is a poor catalyst for the reaction of MgMTP. Lesser enzymatic catalysis in the reaction of MgMTP compared with MgPPi is largely compensated for by the greater intrinsic reactivity of MgMTP. Thus, the larger substrate MgMTP is well accommodated in the active site, and the dissociative reaction of MgMTP is well accommodated in the transition state. The results are interpreted to indicate a dissociative transition state for phosphoryl group transfer by PPi-dependent PFK. A modified synthesis and purification of MTP are described, in which (trimethylsilyl)trifluoromethanesulfonate and tetra-N-butylammonium iodide are used in place of iodotrimethylsilane to dealkylate tetramethyl-MTP.
Halkides
C J
CJ
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
Lightcap
E S
ES
Frey
P A
PA
D000818:Animals; D001667:Binding, Competitive; D011756:Diphosphates / Q000138:chemical synthesis / Q000737:chemistry*; D005636:Fructosephosphates / Q000737:chemistry; D007700:Kinetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011817:Rabbits; D013379:Substrate Specificity
D011756:Diphosphates; D005636:Fructosephosphates; C046010:monothiopyrophosphoric acid; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase