MU-MONOTHIOPYROPHOSPHATE AS A SUBSTRATE FOR INORGANIC PYROPHOSPHATASEAND UDP-GLUCOSE PYROPHOSPHORYLASE

mu-Monothiopyrophosphate (MTP, (O3P)-O-2--S-PO32-) is an excellent sub strate for inorganic pyrophosphatase. The maximum velocity for the hyd rolysis of MgMTP by inorganic pyrophosphatase is 24% of that for MgPPi at pH 8.0 and 5 degrees C and 190% at pH 9.0 and 15 degrees C. The hy drolyses of MnMTP and CoMTP proceed at 24 and greater than or equal to 7%, respectively, of the maximum velocity for the reaction of MgMTP a t pH 8 and 5 degrees C. The maximum velocities for hydrolyses of MnPPi and CoPPi are 31 and 13% of that for MgPPi, respectively. There is no evidence that Mn2+ or Co2+ coordinate with bridging sulfur in MTP in such a way as to affect the rate of hydrolysis. The apparent Michaelis constants at pH 8 and 5 degrees C in the presence of 195 mu M divalen t metal ion are as follows: MgPPi, 12 mu M; MnPPi, 19 mu M; CoPPi, 12 mu M; MgMTP, 45 mu M; MnMTP, 5.3 mu M; and CoMTP, 16 mu M. The apparen t Michaelis constants at pH 9 and 15 degrees C in the presence of 10 m M divalent metal ion are MgPPi, 1.9 mu M and MgMTP, 19.1 mu M. The val ues of k(cat) for MgPPi at pH 8 and 5 degrees C and at pH 9 and 15 deg rees C are 8 s(-1) and 12.4 s(-1), respectively. The values of k(cat) for MgMTP under the same conditions are 2 s(-1) and 24 s(-1), respecti vely. MTP and MgMTP undergo nonenzymatic hydrolysis by a mechanism in which monomeric metaphosphate monoanion (PO3-) is a discrete intermedi ate (Lightcap, E. S., and Prey, P. A. (1992) J. Am. Chem. Soc. 114, 97 50-9755). This reaction is accommodated at the active site of inorgani c pyrophosphatase, indicating that the mechanism of enzymatic hydrolys is is dissociative. MgMTP is also a substrate for UDP-glucose pyrophos phorylase, reacting at 4.8% of the maximum velocity of MgPPi and with a Michaelis constant 17 times larger than that for MgPPi. The P-S bond s of MgMTP are not cleaved in the pyrophosphorylase reaction, but the product UTPbeta,gammaS is chemically unstable and undergoes hydrolysis to UDPbetaS and P-i, making the cleavage of UDP-glucose to glucose-1- P, UDPbetaS and P-i, experimentally irreversible. (C) 1996 Academic Pr ess, Inc.