Kinetically controlled drug resistance: how Penicillium brevicompactum survives mycophenolic acid

The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

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)     

Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP via the covalent E-XMP* intermediate (E-XMP*), with the concomitant reduction of NAD(+). Hydrolysis of E-XMP* is rate-limiting, and the catalytic base required for this step has not been identified. An X-ray crystal structure of Tritrichomonas foetus IMPDH with mizoribine monophosphate (MZP) reveals a novel closed conformation in which a mobile flap occupies the NAD(+)/NADH site [Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., and Hedstrom, L. (2003) Biochemistry 42, 857-863]. In this complex, a water molecule is coordinated between flap residues Arg418 and Tyr419 and MZP in a geometry that resembles the transition state for hydrolysis of E-XMP*, which suggests that the Arg418-Tyr419 dyad activates water. We constructed and characterized two point mutants, Arg418Ala and Tyr419Phe, to probe the role of the Arg418-Tyr419 dyad in the IMPDH reaction. Arg418Ala and Tyr419Phe decrease k(cat) by factors of 500 and 10, respectively, but have no effect on hydride transfer or NADH release. In addition, the mutants display increased solvent isotope effects and increased levels of steady-state accumulation of E-XMP*. Inhibitor analysis indicates that the mutations destabilize the closed conformation, but this effect can account for a decrease in k(cat) of no more than a factor of 2. These observations demonstrate that both the Arg418Ala and Tyr419Phe mutations selectively impair hydrolysis of E-XMP* by disrupting the chemical transformation. Moreover, since the effects of the Tyr419Phe mutation are comparatively small, these experiments suggest that Arg418 acts as the base to activate water.     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

Drug selectivity is determined by coupling across the NAD+ site of IMP dehydrogenase

Drug resistance often results from mutations that are located far from the drug-binding site. The effects of these mutations are perplexing. The inhibition of IMPDH by MPA is an example of this phenomenon. Mycophenolic acid (MPA) is a species-specific inhibitor of IMPDH; mammalian IMPDHs are very sensitive to MPA, while the microbial enzymes are resistant to the inhibitor. MPA traps the covalent intermediate E-XMP and binds in the nicotinamide half of the dinucleotide site. Previous results indicated that about half of the difference in sensitivity derives from residues in the MPA-binding site [Digits, J. A., and Hedstrom, L. (1999) Biochemistry 38, 15388-15397]. The remainder must be attributed to regions outside the MPA-binding site. The adenosine subsite of the NAD+ site is not conserved among IMPDHs and is, therefore, a likely candidate. Our goal is to examine the coupling between the nicotinamide and adenosine sites in order to test this hypothesis. We performed multiple inhibitor experiments with the Tritrichomonas foetus and human type 2 IMPDHs using tiazofurin and ADP, which bind in the nicotinamide and adenosine subsites, respectively. For T. foetus IMPDH, tiazofurin and ADP are extraordinarily synergistic. In contrast, these inhibitors are virtually independent for the human type 2 enzyme. We suggest that the difference in coupling of the nicotinamide and adenosine subsites accounts for the remaining difference in MPA affinity between T. foetus and human IMPDH.     

Is Arg418 the catalytic base required for the hydrolysis step of the IMP dehydrogenase reaction?

The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) catalyzed by IMP dehydrogenase. The reaction involves the reduction of NAD(+) with the formation of a covalent enzyme intermediate (E-XMP). Hydrolysis of E-XMP requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E-XMP is accelerated by the deprotonation of a residue with a pK(a) of approximately 8. A similar deprotonation stabilizes the closed conformation; this residue has a pK(a) of >or=6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of either the hydrolysis of E-XMP or the conformational change, which suggests that Tyr419 is not the residue that activates water. In contrast, the conformational change becomes pH-independent when Arg418 is substituted with Gln. Lys can replace the function of Arg418 in the hydrolysis reaction but does not stabilize the closed conformation. The simplest explanation for these observations is that Arg418 serves as the base that activates water in the IMPDH reaction.     

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.     

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.     

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].     

Adaptive evolution of drug targets in producer and non-producer organisms

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance. Surprisingly, all members of the subgenus Penicillium contain genes encoding IMPDHs of both the A and B types, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred before and independently of the acquisition of the MPAbiosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant, whereas the IMPDHs from a non-producer are MPA-sensitive. Resistance comes with a catalytic cost: whereas P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its kcat/Km value is 0.5% of 'normal'. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA-binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in the subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.     

The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase

Mizoribine monophosphate (MZP) is the active metabolite of the immunosuppressive agent mizoribine and a potent inhibitor of IMP dehydrogenase (IMPDH). This enzyme catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD via a covalent intermediate at Cys319 (E-XMP). Surprisingly, mutational analysis indicates that MZP is a transition state analogue although its structure does not resemble that of the expected transition state. Here we report the X-ray crystal structure of the E.MZP complex at 2.0 A resolution that reveals a transition state-like structure and solves the mechanistic puzzle of the IMPDH reaction. The protein assumes a new conformation where a flap folds into the NAD site and MZP, Cys319, and a water molecule are arranged in a geometry resembling the transition state. The water appears to be activated by interactions with a conserved Arg418-Tyr419 dyad. Mutagenesis experiments confirm that this new closed conformation is required for the hydrolysis of E-XMP, but not for the reduction of NAD. The closed conformation provides a structural explanation for the differences in drug selectivity and catalytic efficiency of IMPDH isozymes.     

Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

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.     

Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties

Inosine-5′-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed.     

Molecular genetic study of autosomal dominant retinitis pigmentosa in Lithuanian patients

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene - Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene - Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis.

The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.