Monovalent Cation Activation and Kinetic Mechanism of Inosine 5′-Monophosphate Dehydrogenase

Abstract

Human type II inosine 5-monophosphate dehydrogenase has been purified to homogeneity from an Escherichia coli strain that express large quantities of the enzyme from the cloned gene. which is the rate-limiting step in the biosynthesis of guanine nucleotides (1, 2). IMPDH activity has been linked to malignancy because enzyme levels are greatly elevated in tumor tissues and the levels are correlated with cell growth rates (3-5). There are two isozymes of human IMPDH, denoted types I and II (or IMPDH-h1 and IMPDH-h2) (6). IMPDH-h2 expression is selectively up-regulated in leukemias and brain tumor tissues (7,8). Based on these observations, IMPDH has been recognized as a target for antitumor agents (9).Isoforms of IMPDH have been purified from a variety of eukaryotic and prokaryotic sources; all are tetrameric with subunit molecular weights of ϳ56 kDa (10 -14). Both IMPDH-h1 and IMPDH-h2 subunits have 514 amino acids, and they have 84% sequence identity (6). IMPDH-h2 is inactivated by 6-chloro-purine ribotide, which reacts with Cys-331 in the IMP binding site, providing the only evidence for the location of the IMP binding site (6,15,16 , is required for maximal activity, but the mechanism and extent of the activation are unknown. Monovalent cations of different radii show different abilities to activate the enzymes from Bacillus subtilis and sarcoma 180 cells (17,18). An absolute requirement for a monovalent cation activator has not yet been demonstrated, perhaps due to the use of sodium or potassium salts of substrates or other components in assay solutions, and thus 10% or more of the maximum activities have been reported in experiments in which no monovalent cations were intentionally added (17, 18).A steady state ordered sequential Bi Bi mechanism in which IMP binds before NAD and XMP is released after NADH is commonly used to describe the IMPDH-catalyzed reaction (19). Because this mechanism does not include the monovalent cation, it is not complete. Another partially random rapid equilibrium mechanism including K ϩ , IMP, and NAD was proposed by Morrison and co-workers (20) for the enzyme from Aerobacter aerogenes. In this mechanism, K ϩ and IMP are proposed to bind randomly to the enzyme, whereas NAD does not bind unless K ϩ or both K ϩ and IMP are bound. In this study, we constructed an Escherichia coli expression system for IMPDH-h2 and purified the recombinant protein to homogeneity. The activation of the recombinant IMPDH-h2 by various monovalent cations has been characterized and a larger than 100-fold activation by a monovalent cation, such as K ϩ , was found. A steady state kinetic mechanism including the monovalent cation activation of IMPDH-h2 is proposed based on initial velocity and product inhibition studies. Substrate and product equilibrium binding experiments were performed to directly determine binding affinities and to provide further evidence supporting the proposed mechanism.EXPERIMENTAL PROCEDURES NAD, NADH, alkaline phosphatase, and the free acid of IMP were purchased from Sigma. The so...

The Conformation of Inosine 5′-Monophosphate (IMP) Bound to IMP Dehydrogenase Determined by Transferred Nuclear Overhauser Effect Spectroscopy

“…Our previous work showed that there were four IMP-binding sites per 223-kDa IMPDH-h2 tetramer (8). In other studies, the IMP-binding site was shown to be near Cys-331 (16).…”

“…The steady state kinetic mechanism of the enzyme from various sources has been shown to be steady state ordered sequential Bi Bi in which IMP binds before NAD, and XMP is released before NADH (3,(5)(6)(7). Our previous study of the human tumor form of IMP dehydrogenase (human type II IMPDH or IMPDH-h2) extended this mechanism by analysis of the activation of the enzyme by various monovalent cations and proposed a steady state mechanism including the monovalent cation activator (8). In this mechanism, the enzyme binds K ϩ first, IMP second, and then NAD; the product NADH is released before XMP.…”

Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

“…the conversion of XMP to IMP. Product inhibition of the forward reaction by XMP and NADH is characteristic of several IMPDH enzymes studied to date (Verham et al, 1987;Xiang et al, 1996). The Mt-GuaB2 enzyme exhibited substrate inhibition in vitro at high concentrations of NADH and XMP.…”

Conformational Changes and Stabilization of Inosine 5′-Monophosphate Dehydrogenase Associated with Ligand Binding and Inhibition by Mycophenolic Acid

“…All possess a similar homotetrameric quaternary arrangement of about 55-57-kDa subunits, and the numerous enzyme kinetic characterizations (reviewed recently by Wu (19) and (25)) suggest a common mechanism that is unusual among dehydrogenases. In most other dehydrogenases substrate binding is either random or the hydride acceptor binds first (26).…”