George D. Markham

Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

Crystal Structure of S-Adenosylmethionine Synthetase

The structure of S-adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6.) from Escherichia coli has been determined at 3.0 Å resolution by multiple isomorphous replacement using a uranium derivative and the selenomethionine form of the enzyme (SeMAT). The SeMAT data (9 selenomethionine residues out of 383 amino acid residues) have been found to have a sufficient phasing power to determine the structure of the 42,000 molecular weight protein by combining them with the other heavy atom derivative data (multiple isomorphous replacement). The enzyme consists of four identical subunits; two subunits form a spherical tight dimer, and pairs of these dimers form a peanut-shaped tetrameric enzyme. Each pair dimer has two active sites which are located between the subunits. Each subunit consists of three domains that are related to each other by pseudo-3-fold symmetry. The essential divalent (Mg/Co) and monovalent (K) metal ions and one of the product, P ions, were found in the active site from three separate structures.

EPR of Mn(II) Complexes with Enzymes and Other Proteins

The history of EPR applications to the study of manganese dates to the very first successful resonance experiments by Zavoisky (1945). Since then there have been numerous EPR studies of Mn(II) in diverse materials. The reader may find references to many of these studies in review articles and monographs on EPR (Abragam and Bleaney, 1970; Goodman and Raynor, 1970; Konig, 1968; Kaiser and Kevan, 1968). Applications of EPR to studies of Mn(II) complexes with proteins began with studies by Malmstrom et al. (1958) wherein the strong isotropic EPR signal for “unbound” Mn(H2O) 6 2+ was used to determine dissociation constants for Mn(II)-protein complexes. Such analytical applications of the EPR signal for Mn(H2O) 6 2+ (Cohn and Townsend, 1954) for measuring dissociation constants have continued. However, during the last decade the EPR signals for the protein-bound Mn(II) have been measured for enzymes and other proteins, and this latter application is the basis for the present chapter.

Structure-function relationships in methionine adenosyltransferases

Abstract.Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.

Methylthioadenosine Phosphorylase Regulates Ornithine Decarboxylase by Production of Downstream Metabolites

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639–6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1Δ cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.

The Bifunctional Active Site of S-Adenosylmethionine Synthetase ROLES OF THE ACTIVE SITE ASPARTATES

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPPi) from ATP, and subsequently PPPi is hydrolyzed to PPi and Pibefore product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg2+ ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k cat reductions of ∼103-fold in AdoMet synthesis, whereas the MgATP and methionine K m values change by less than 3- and 8-fold, respectively. In the partial reaction of PPPihydrolysis, mutants of the Mg2+ binding residues have >700-fold reduced catalytic efficiency (k cat/K m ), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPPi hydrolysis by Mg2+ site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPPi hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca2+ uniquely activates the D271A mutant enzyme to 15% of the level of Mg2+, in contrast to the ∼1% Ca2+ activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca2+ and the smaller Mg2+.

Shape Shifting Leads to Small-Molecule Allosteric Drug Discovery

Enzymes that regulate their activity by modulating an equilibrium of alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) constitute a recently described mode of allostery. The oligomeric equilibrium for porphobilinogen synthase (PBGS) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. A phylogenetically diverse allosteric site specific to hexamers is proposed as an inhibitor binding site. Inhibitor binding is predicted to draw the oligomeric equilibrium toward the low-activity hexamer. In silico docking enriched a selection from a small-molecule library for compounds predicted to bind to this allosteric site. In vitro testing of selected compounds identified one compound whose inhibition mechanism is species-specific conversion of PBGS octamers to hexamers. We propose that this strategy for inhibitor discovery can be applied to other proteins that use the morpheein model for allosteric regulation.

The CBS subdomain of inosine 5’-monophosphate dehydrogenase regulates purine nucleotide turnover

Inosine 5′‐monophosphate dehydrogenase (IMPDH) catalyses the rate‐limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine‐prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild‐type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14‐C tracers and 33‐P pulse‐chasing revealed mutation‐associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli.

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

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. Steady state kinetic studies have been used to characterize the activation by monovalent cations, including Li, Na, K, Rb, Cs, Tl, NH, and N(CH). The enzyme has less than 1% of the maximal activity in the absence of an added monovalent cation, such as K, Na, Rb, Tl, or NH. The enzyme is activated by K and Tl at lower concentrations than those of other monovalent cations. Li and N(CH) do not activate the enzyme, nor do they inhibit the K-activated enzyme, implying that ionic radius is important in binding selectivity. The K values for both substrates and V differ with different monovalent cations. Initial velocity and product inhibition kinetic data are consistent with an ordered steady state mechanism in which the enzyme binds K first, IMP second, and then NAD; the product NADH is released before xanthosine 5′-monophosphate. Substrate and product binding experiments support this mechanism and show the presence of one substrate binding site per subunit. Several rate constants were obtained from a computer simulation of the complete steady state rate equation.

Enzymatic Properties of S-Adenosylmethionine Synthetase from the Archaeon Methanococcus jannaschii

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent. MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes. In this study the functional properties of purified recombinant MjMAT have been evaluated. The products of the reaction are AdoMet, PPi, and Pi; >90% of the Pioriginates from the γ-phosphoryl group of ATP. The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology. The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PPi and Pi. The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety. MjMAT has activity at 70 °C comparable with that of EcMAT at 37 °C, consistent with the higher temperature habitat of M. jannaschii. The activation energy for AdoMet formation is larger than that for the E. coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts. The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs.