Asp338 controls hydride transfer in Escherichia coli IMP dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+). This reaction involves the formation of a covalent adduct with an active site Cys. This intermediate, E-XMP, hydrolyzes to produce XMP. The mutation of Asp338 to Ala severely impairs the activity of Escherichia coli IMPDH, decreasing the value of k(cat) by 650-fold. No (D)V(m) or (D)V/K(m) isotope effects are observed when 2-(2)H-IMP is the substrate for wild-type IMPDH. Values of (D)V(m) = 2.6 and (D)V/K(m) (IMP) = 3.4 are observed for Asp338Ala. Moreover, while a burst of NADH production is observed for wild-type IMPDH, no burst is observed for Asp338Ala. These observations indicate that the mutation has decreased the rate of hydride transfer by at least 5 x 10(3)-fold. In contrast, k(cat) for the hydrolysis of 2-chloroinosine-5'-monophosphate is decreased by only 8-fold. In addition, the rate constant for inactivation by 6-chloropurine riboside 5'-monophosphate is increased by 3-fold. These observations suggest that the mutation has little effect on the nucleophilicity of the active site Cys residue. These results are consistent with a recent crystal structure that shows a hydrogen bonding network between Asp338, the 2'-OH of IMP, and the amide group of NAD(+) [Colby, T. D., Vanderveen, K., Strickler, M. D., Markham, G. D., and Goldstein, B. M. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3531-3536].     

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of a ternary complex of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: NAD+ orients the active site loop for catalysis

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to XMP with the reduction of NAD(+), which is the rate-limiting step in the biosynthesis of guanine nucleotides. IMPDH is a promising target for chemotherapy. Microbial IMPDHs differ from mammalian enzymes in their lower affinity for inhibitors such as mycophenolic acid (MPA) and thiazole-4-carboxamide adenine dinucleotide (TAD). Part of this resistance is determined by the coupling between nicotinamide and adenosine subsites in the NAD(+) binding site that is postulated to involve an active site flap. To understand the structural basis of the drug selectivity, we solved the X-ray crystal structure of the catalytic core domain of Tritrichomonas foetus IMPDH in complex with IMP and beta-methylene-TAD at 2.2 A resolution. Unlike previous structures of this enzyme, the active site loop is ordered in this complex, and the catalytic Cys319 is 3.6 A from IMP, in the same plane as the hypoxanthine ring. The active site loop forms hydrogen bonds to the carboxamide of beta-Me-TAD which suggests that NAD(+) promotes the nucleophillic attack of Cys319 on IMP. The interactions of the adenosine end of TAD are very different from those in the human enzyme, suggesting the NAD(+) site may be an exploitable target for the design of antimicrobial drugs. In addition, a new K(+) site is observed at the subunit interface. This site is adjacent to beta-Me-TAD, consistent with the link between the K(+) activation and NAD(+). However, contrary to the coupling model, the flap does not cover the adenosine subsite and remains largely disordered.     

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.     

Importance of Glu(282) in transmembrane segment M3 of the Na(+),K(+)-ATPase for control of cation interaction and conformational changes

Glu(282) located in the NH(2)-terminal part of transmembrane helix M3 of the Na(+),K(+)-ATPase was replaced by alanine, glycine, leucine, lysine, aspartate, or glutamine, and the effects of the mutations on the overall and partial reactions of the enzyme were analyzed. The mutations affected at least 3 important functions of the Na(+),K(+)-ATPase: (i) the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme, (ii) Na(+) binding at the cytoplasmically facing sites of E(1), and (iii) long-range interaction controlling dephosphorylation. In mutants Glu(282) --> Lys and Glu(282) --> Asp, the E(1) form was favored during ATP hydrolysis, whereas the E(2) form was favored in Glu(282) --> Ala and Glu(282) --> Gly. Regardless of the change of conformational equilibrium, all the mutants displayed a reduced apparent affinity for Na(+), at least 3-fold for Glu(282) --> Lys and Glu(282) --> Asp, suggesting a direct effect on the Na(+) binding properties of E(1). Glu(282) --> Ala and Glu(282) --> Gly exhibited an extraordinary high rate of ATP hydrolysis in the mere presence of Na(+) without K(+) ("Na(+)-ATPase activity"), because of an increased rate of dephosphorylation of E(2)P. These results are in accordance with the hypothesis that Glu(282) is involved in the communication between the cation binding pocket and the catalytic site and in control of the cytoplasmic entry pathway for Na(+).     

Allosteric activation via kinetic control: potassium accelerates a conformational change in IMP dehydrogenase

Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K(+) activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K(+) dependence of k(cat) derives from the rate of flap closure, which increases by ≥65-fold in the presence of K(+). We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K(+) accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K(+), validating the computational models. When K(+) is replaced with a dummy ion, the residues of the K(+) binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K(+) mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K(+) changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.     

Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.     

Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid

IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.     

Residue Asp-189 controls both substrate binding and the monovalent cation specificity of thrombin

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na(+). The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na(+)-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na(+) to the smaller cation Li(+) or to the larger cation Rb(+), respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na(+) (Asp-189) to Li(+) (Ser-189) or Rb(+) (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.     

Acid-base catalysis in the chemical mechanism of inosine monophosphate dehydrogenase

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K+-dependent reaction IMP + NAD + H2O --> XMP + NADH + H+ which is the rate-limiting step in guanine nucleotide biosynthesis. The catalytic mechanism of the human type-II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, of the hydrolysis of 2-chloro-IMP (which yields XMP and Cl- in the absence of NAD), and of inactivation by the affinity label 6-chloro-purine-ribotide (6-Cl-PRT). The pH dependence of the IMPDH reaction shows bell-shaped profiles for kcat and the kcat/Km values for both IMP and NAD, illustrating the involvement of both acidic and basic groups in catalysis. Half-maximal kcat values occur at pH values of 7.2 and 9.8; similar pK values of 6.9 and 9.4 are seen in the kcat/Km profile for NAD. The kcat/Km profile for IMP, which binds first in the predominantly ordered kinetic mechanism, shows pK values of 8.1 and 7.3 for acidic and basic groups, respectively. None of the kinetic pK values correspond to ionizations of the free substrates and thus reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the active site sulfhydryl of cysteine-331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E.6-Cl-PRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl which adds to C-2 of IMP prior to NAD reduction. The kcat and kcat/Km values for hydrolysis of 2-Cl-IMP show a pK value of 9.9 for a basic group, similar to that seen in the overall reaction, but do not exhibit the ionization of an acidic group. Surprisingly, the rates of 2-Cl-IMP hydrolysis and of inactivation by 6-Cl-PRT are not stimulated by K+, in contrast to the >100-fold K+ activation of the IMPDH reaction. Apparently the enigmatic role of K+ lies in the NAD(H)-dependent segment of the IMPDH reaction. To evaluate the importance of hydrogen bonding in substrate binding, several deamino- and deoxy-analogues of IMP were tested as substrates and inhibitors. Only 2'-deoxy-IMP was a substrate; the other compounds tested were competitive inhibitors with Ki values at most 10-fold greater than the KD for IMP, illustrating the greater importance of hydrogen-bonding interactions in the chemistry of the IMPDH reaction than simply in nucleotide binding.     

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Crystal structure at 2.4 A resolution of Borrelia burgdorferi inosine 5'-monophosphate dehydrogenase: evidence of a substrate-induced hinged-lid motion by loop 6

The conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) is the committed and rate-limiting reaction in de novo guanine nucleotide biosynthesis. Inosine 5'- monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the oxidation of IMP to XMP with the concomitant reduction of nicotinamide adenine dinucleotide (from NAD(+) to NADH). Because of its critical role in purine biosynthesis, IMPDH is a drug design target for anticancer, antiinfective, and immunosuppressive chemotherapy. We have determined the crystal structure of IMPDH from Borrelia burgdorferi, the bacterial spirochete that causes Lyme disease, with a sulfate ion bound in the IMP phosphate binding site. This is the first structure of IMPDH in the absence of substrate or cofactor where the active-site loop (loop 6), which contains the essential catalytic residue Cys 229, is clearly defined in the electron density. We report that a seven residue region of loop 6, including Cys229, is tilted more than 6 A away from its position in substrate- or substrate analogue-bound structures of IMPDH, suggestive of a conformational change. The location of this loop between beta6 and alpha6 links IMPDH to a family of beta/alpha barrel enzymes known to utilize this loop as a functional lid during catalysis. Least-squares minimization, root-mean-square deviation analysis, and inspection of the molecular surface of the loop 6 region in the substrate-free B. burgdorferi IMPDH and XMP-bound Chinese hamster IMPDH show that loop 6 follows a similar pattern of hinged rigid-body motion and indicates that IMPDH may be using loop 6 to bind and sequester substrate and to recruit an essential catalytic residue.     

Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.     

The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.     

Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).     

A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.     

An isothermal titration calorimetry study of the binding of substrates and ligands to the tartrate dehydrogenase from Pseudomonas putida reveals half-of-the-sites reactivity

An isothermal titration calorimetric study of the binding of substrates and inhibitors to different complexes of tartrate dehydrogenase (TDH) from Pseudomonas putida was carried out. TDH catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent oxidative decarboxylation of d-malate and has an absolute requirement for both a divalent and monovalent metal ion for activity. The ligands Mn(2+), meso-tartrate, oxalate, and reduced nicotinamide adenine dinucleotide (NADH) bound to all TDH complexes with a stoichiometry of 1 per enzyme dimer. The exception is NAD, which binds to E/K(+), E/K(+)/Mn(2+), and E/K(+)/Mg(2+) complexes with a stoichiometry of two per enzyme dimer. The binding studies suggest a half-of-the-sites mechanism for TDH. No significant heat changes were observed for d-malate in the presence of the E/K(+)/Mn(2+) complex, suggesting that it did not bind. In contrast, meso-tartrate does bind to E/K(+)/Mn(2+) but gives no significant heat change in the presence of E/Mn(2+), suggesting that K(+) is required for meso-tartrate binding. meso-Tartrate also binds with a large DeltaC(p) value and likely binds via a different binding mode than d-malate, which binds only in the presence of NAD. In contrast to all of the other ligands tested, the binding of Mn(2+) is entropically driven, likely the result of the entropically favored disruption of ordered water molecules coordinated to Mn(2+) in solution that are lost upon binding to the enzyme. Oxalate, a competitive inhibitor of malate, binds with the greatest affinity to E/K(+)/Mn(2+)/NADH, and its binding is associated with the uptake of a proton. Overall, with d-malate as the substrate, data are consistent with a random addition of K(+), Mn(2+), and NAD followed by the ordered addition of d-malate; there is significant synergism in the binding of NAD and K(+). Although the binding of meso-tartrate also requires enzyme-bound K(+) and Mn(2+), the binding of meso-tartrate and NAD is random.     

Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR.