Studies on inosine monophosphate dehydrogenase. Steady state kinetics

Heyde, Elizabeth; Nagabhushanam, A.; Vonarx, Margaret; Morrison, John F.

doi:10.1016/0005-2744(76)90314-4

Cited by 33 publications

(29 citation statements)

References 14 publications

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“…4B) and replotting these values against IMP concentration. These data were fit to the Michaelis-Menten equation: K m (IMP) ϭ 29 Ϯ 8 M. These values are comparable to those reported for other bacterial IMPDHs (22,26). Thus B. burgdorferi guaB encodes an IMPDH with typical kinetic properties.…”

Section: Resultssupporting

confidence: 72%

Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

Zhou¹,

Cahoon²,

Rosa³

et al. 1997

Journal of Biological Chemistry

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Inosine 5-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. IMPDH converts IMP to xanthosine 5-monophosphate with concomitant conversion of NAD ؉ to NADH. All IMPDHs characterized to date contain a 130-residue "subdomain" that extends from an N-terminal loop of the ␣/␤ barrel domain. Inosine 5Ј-monophosphate dehydrogenase catalyzes the conversion of IMP to XMP 1 with the concomitant reduction of NAD to NADH. This reaction is the rate-limiting step in guanine nucleotide biosynthesis, and is therefore a target for numerous chemotherapeutic agents (1). IMPDH inhibitors are used clinically in antiviral (ribavirin) and immunosuppressive therapies (mycophenolate mofetil and mizoribine) (2-4). In addition, IMPDH inhibitors have anti-tumor and antibiotic activity (5, 6). Mammalian and bacterial IMPDHs have significantly different kinetic properties and inhibitor sensitivities, which suggests that species-specific IMPDH inhibitors can be developed that will be useful in treating bacterial and parasitic infections (7-9). Indeed, many studies have shown that purine metabolism plays an important role in bacterial virulence (10 -13).The spirochete Borrelia burgdorferi is the causative agent of Lyme disease (14). This disease is transmitted by ticks of the Ixodes ricinus complex and is found worldwide. The genes encoding GMP synthase (guaA) and IMPDH (guaB) are located on a 26-kb circular plasmid (cp26) in B. burgdorferi (15). Genes carried by plasmids generally confer selective advantage in a particular environmental niche. The unique plasmid location of these housekeeping genes in B. burgdorferi may be related to their role in the transmission cycle between ticks and mammals. In ticks, guanine is the major nitrogenous waste product and therefore accumulates to high levels. However, in mammals, purine levels are low and limiting for bacterial growth. Therefore expression of the gua genes would be unnecessary for the survival of B. burgdorferi in ticks, but critical for survival in a mammalian host. Consistent with an adaptive role of cp26 in the mammalian environment, the gua genes are linked with ospC, which is induced during tick feeding. ospC encodes a protein that appears on the outer surface of the spirochete immediately preceding transmission to the mammal (16).The identity of B. burgdorferi guaA was confirmed by complementation of GMP synthase-deficient Escherichia coli (15); however, the guaB homolog did not complement IMPDHdeficient E. coli. The failure to observe complementation by B. burgdorferi guaB most likely results from incompatible promoters or unstable protein. Alternatively, the guaB homolog may not encode an active IMPDH. Indeed, the guaB homolog, with a predicted molecular mass of 44 kDa, is 10 kDa smaller than typical IMPDHs. This difference in size results from the loss of 130 residues (residues 110 -244, Chinese hamster IMPDH numbering) in the middle of IMPDH. These residues have been replaced with 50 residues of unrelated sequence (15).The cry...

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Section: Resultssupporting

confidence: 72%

Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

Zhou¹,

Cahoon²,

Rosa³

et al. 1997

Journal of Biological Chemistry

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“…The kinetic data shown in Figs. 3 and 4 are not consistent with the partially random rapid equilibrium reaction mechanism suggested by Morrison and co-workers (20) for IMPDH from A. aerogenes because the common intersection (Fig. 3E) in plots of 1/V versus 1/[NAD] at different concentrations of K ϩ is not on the vertical axis.…”

Section: Discussionmentioning

confidence: 42%

“…4C) and the characteristic off-axis common intersections in Figs. 3 (D and F) and 4 (D, E, and F) are the patterns predicted by the steady state ordered sequential Bi Bi mechanism in which IMP binds first and XMP is released last (20). This part of the mechanism has been proposed by other workers (13, 19, 28 -32).…”

Section: Discussionmentioning

confidence: 90%

“…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.…”

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confidence: 99%

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

Xiang

Taylor

Markham

1996

Journal of Biological Chemistry

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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...

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“…However, our kinetic data show that K ϩ enhances the affinity constants for ADP-Mg In addition to its effect on PK, K ϩ is an activator of various other enzymes. In this context, it is particularly relevant that K ϩ activates inosine 5Ј-monophosphate dehydrogenase from Aerobacter aerogenes by changing its kinetic mechanism from random order to ordered with inosine monophosphate and NAD ϩ as the first and second substrate, respectively (44,45). The authors suggest that K ϩ induces a conformational change that permits the binding of NAD ϩ (44,45).…”

Section: The Effect Of K ϩ and Ligands On The Structure Of Pyruvate Kmentioning

confidence: 99%

Pyruvate Kinase Revisited

Oria-Hernández

Cabrera

Pérez‐Montfort

et al. 2005

Journal of Biological Chemistry

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Studies on inosine monophosphate dehydrogenase. Steady state kinetics

Cited by 33 publications

References 14 publications

Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

Expression, Purification, and Characterization of Inosine 5′-Monophosphate Dehydrogenase from Borrelia burgdorferi

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

Pyruvate Kinase Revisited

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