mu-Monothiopyrophosphate as a substrate for inorganic pyrophosphatase and UDP-glucose pyrophosphorylase.

mu-Monothiopyrophosphate (MTP, (2-)O3P-S-PO3(-2)) is an excellent substrate for inorganic pyrophosphatase. The maximum velocity for the hydrolysis 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 hydrolyses of MnMTP and CoMTP proceed at 24 and > or = 7%, respectively, of the maximum velocity for the reaction of MgMTP at 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 microM divalent metal ion are as follows: MgPPi, 12 microM; MnPPi, 19 microM; CoPPi, 12 microM; MgMTP, 45 microM; MnMTP, 5.3 microM; and CoMTP, 16 microM. The apparent Michaelis constants at pH 9 and 15 degrees C in the presence of 10 mM divalent metal ion are MgPPi, 1.9 microM and MgMTP, 19.1 microM. The values of kcat for MgPPi at pH 8 and 5 degrees C and at pH 9 and 15 degrees C are 8 s(-1) and 12.4 s(-1), respectively. The values of kcat for MgMTP under the same conditions are 2 s(-1) and 24 s(-1), respectively. MTP and MgMTP undergo nonenzymatic hydrolysis by a mechanism in which monomeric metaphosphate monoanion (PO3) is a discrete intermediate (Lightcap, E.S., and Frey, P.A. (1992) J. Am. Chem. Soc. 114, 9750-9755). This reaction is accommodated at the active site of inorganic pyrophosphatase, indicating that the mechanism of enzymatic hydrolysis is dissociative. MgMTP is also a substrate for UDP-glucose pyrophosphorylase, 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 bonds of MgMTP are not cleaved in the pyrophosphorylase reaction, but the product UTP beta gamma S is chemically unstable and undergoes hydrolysis to UDP beta S and Pi, making the cleavage of UDP-glucose to glucose-1-P, UDP beta S and Pi, experimentally irreversible.