μ-Monothiopyrophosphate as a Substrate for Inorganic Pyrophosphatase and UDP-Glucose Pyrophosphorylase

μ-Monothiopyrophosphate (MTP,2−O3P–S–PO2−3) is an excellent substrate for inorganic pyrophosphatase. The maximum velocity for the hydrolysis of MgMTP by inorganic pyrophosphatase is 24% of that for MgPPiat pH 8.0 and 5°C and 190% at pH 9.0 and 15°C. The hydrolyses of MnMTP and CoMTP proceed at 24 and ≥7%, respectively, of the maximum velocity for the reaction of MgMTP at pH 8 and 5°C. The maximum velocities for hydrolyses of MnPPiand CoPPiare 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°C in the presence of 195 μMdivalent metal ion are as follows: MgPPi, 12 μM; MnPPi, 19 μM; CoPPi, 12 μM; MgMTP, 45 μM; MnMTP, 5.3 μM; and CoMTP, 16 μM. The apparent Michaelis constants at pH 9 and 15°C in the presence of 10 mMdivalent metal ion are MgPPi, 1.9 μMand MgMTP, 19.1 μM. The values ofkcatfor MgPPiat pH 8 and 5°C and at pH 9 and 15°C are 8 s−1and 12.4 s−1, respectively. The values ofkcatfor MgMTP under the same conditions are 2 s−1and 24 s−1, respectively. MTP and MgMTP undergo nonenzymatic hydrolysis by a mechanism in which monomeric metaphosphate monoanion (PO−3) 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 MgPPiand 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 UTPβ,γS is chemically unstable and undergoes hydrolysis to UDPβS and Pi, making the cleavage of UDP-glucose to glucose-1-P, UDPβS and Pi, experimentally irreversible.