Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax.

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei.

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial import sequence at its N-terminus and has a variant IPC motif replacing the second VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cytoplasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are expressed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclonal antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibody to TRYP2 were generated in mice. In-situ immunofluorescence and immunoelectron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol, whereas TRYP2 was principally localised in the mitochondrion.

Pattern of ophthalmological accidents and emergencies presenting to hospitals.

STUDY OBJECTIVE: To investigate the numbers and characteristics of patients with ophthalmological accidents and emergencies presenting to hospitals. DESIGN: Prospective survey over eight weeks. SETTING: Two general and one ophthalmic accident and emergency departments, two general outpatient departments, and an eye hospital ward consulting room (all in two teaching hospitals) in Newcastle upon Tyne. MEASUREMENTS AND MAIN RESULTS: Consultation numbers by age, sex, health district of residence, source of referral, diagnosis, and disposal were determined. An average of 37 ophthalmological emergency patients were seen daily. The all cause consultation rate per 1000 population for Newcastle residents was 2.64 (17.2 per year); for injuries it was 1.10 (7.2 per year) and for inflammations the rate was 0.91 (5.9 per year). Consultation rates per 1000 were 3.5 for males and 1.8 for females, the excess being explained by the higher risk of injury to men. Most patients were self-referred (58%), consulted during office hours (79.6%), were attended by senior house officers working alone (83.9%), and were asked to return for follow up (66.1%). Patients in an accident and emergency department seldom saw a consultant in their initial management. The diagnoses of patients from outside Newcastle were little different from those who lived within the city. The 10 commonest problems accounted for 68% of all cases. Injuries were the commonest problem (40.9% of all diagnoses). CONCLUSION: Ophthalmological accident and emergencies are an important component of an accident and emergency department workload. These patients are usually seen by junior doctors, some untrained in ophthalmology. The wide range of presenting problems poses a challenge for training and the organisation of effective referral chains, while the gender difference in injury rates points to the potential for prevention.

Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei. Purification, specificity, and kinetic mechanism.

Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Trypanosoma brucei brucei represents approximately 0.2% of the soluble protein in this organism. The enzyme has been purified over 400-fold to >95% homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of Mr 36,000 subunits. The kcat/Km for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x 10(6), and 0.83 x 10(6) M -1 s-1, respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degrees C is 34 s-1. Pyrimidine nucleosides are poor substrates with kcat/Km values of approximately 10(3) M -1 s-1. Deoxynucleosides are also poor substrates with kcat/Km values near 10(2) M -1 s-1. AMP is not a detectable substrate and there is no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin), and formycin B are competitive inhibitors with Kis of 1.8, 59, and 13 microM, respectively. The Km shows a slight dependence on pH with a pH optimum around 7. The Vmax/Km data indicate there are two ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The Vmax data indicate three ionizable groups involved in catalysis. Two essential groups exhibit pKa values of 8.8, and a third group with a pKa of 6.5 increases the Vmax an additional 10-fold. All three groups must be protonated for optimum catalytic activity.

Catalytic and allosteric mechanism of AMP nucleosidase from primary, beta-secondary, and multiple heavy atom kinetic isotope effects.

Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase [DeWolf, W. E., Jr., Emig, F. A., & Schramm, V. L. (1986) Biochemistry 25, 4132-4140] indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Changes in kinetic isotope effects occurred as a function of allosteric activator, demonstrating that allosteric activation alters transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as [V/K(normal isotope]/[V/K(heavy isotope)], were observed with [2'-2H]AMP (1.061 +/- 0.002), [9-15N]AMP (1.030 +/- 0.003), [1'-2H]AMP (1.045 +/- 0.002), and [1'-14C]AMP (1.035 +/- 0.002) when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the [2'-2H]AMP effect (1.043 +/- 0.002) and the [1'-2H]AMP effect (1.030 +/- 0.003) and caused a smaller decrease of the 14C and 15N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects to 1.084 +/- 0.004 for [1'-2H,1'-14C]AMP and to 1.058 +/- 0.002 for [9-15N,1'-14C]AMP with the enzyme in the absence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of allosteric activation on the primary and secondary kinetic isotope effects for three AMP nucleosidases.

Kinetic isotope effects (V/K) were measured with AMP nucleosidases isolated from Azotobacter vinelandii, from a Vmax mutant enzyme of A. vinelandii and from Escherichia coli. Specifically labeled AMP substrates were used to measure 3H secondary and 14C primary kinetic isotope effects on the N-glycosidic bond hydrolysis of AMP in the presence and absence of the allosteric activator, MgATP. Use of the three enzymes, variable MgATP concentration, a poor substrate (dAMP), and variable pH has allowed determination of the isotope effects over a 5000-fold range in the catalytic turnover number. The primary kinetic isotope effects were 1.025 +/- 0.004 and 1.041 +/- 0.006 for the native A. vinelandii enzyme and mutant enzyme, respectively, and were independent of MgATP concentration. The E. coli AMP nucleosidase had a primary isotope effect of 1.019 +/- 0.003 which was also independent of MgATP concentration. The secondary kinetic isotope effect decreased from 1.066 +/- 0.003 to 1.045 +/- 0.002 for the native enzyme from A. vinelandii as the concentration of MgATP increased from 0 to 500 microM. The secondary isotope effect of the mutant enzyme remained constant at 1.088 +/- 0.005 as the MgATP concentration increased from 0 to 500 microM. The secondary isotope effect of the E. coli enzyme showed a similar pattern to that of the native enzyme, decreasing from 1.087 +/- 0.003 to 1.050 +/- 0.003 as the enzyme was saturated with MgATP at a constant concentration of AMP. Saturation with AMP in the absence of MgATP gave similar results and suggested that AMP can cause the allosteric transition. Both the primary and secondary isotope effects for the native enzyme from A. vinelandii remained constant as the pH was varied in the absence of MgATP. Secondary isotope effects with a poor substrate, dAMP, were 1.08 for both the mutant and wild type enzymes from A. vinelandii in the presence of allosteric activator. In the native enzyme, this isotope effect was independent of MgATP concentration. The relative insensitivity in the magnitude of observed isotope effects to pH, allosteric activator, the mutant enzyme, and a poor substrate (dAMP) indicate that intrinsic isotope effects are being expressed. The data are interpreted in terms of a single rate-limiting transition state for hydrolysis of the N-glycosidic bond, although other mechanisms cannot be eliminated. Using this model, the transition states of the native A. vinelandii and E. coli enzymes exhibit properties of both dissociative and associative mechanisms but become more associative as the allosteric activator becomes saturating.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding modes for substrate and a proposed transition-state analogue of protozoan nucleoside hydrolase.

The transition-state structure for inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from Crithidia fasciculata is characterized by oxycarbonium character in the ribosyl and weak bonds to the departing hypoxanthine and incipient water nucleophile [Horenstein, B. A., Parkin, D. W., Estupiñán, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. Inhibitors designed to resemble the transition state are slow-onset, tight-binding inhibitors with observed Km/Ki values up to 2 x 10(5) [Schramm, V. L., Horenstein, B. H., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259-18262]. Although slow-onset, tight binding is consistent with transition-state stabilization, more direct evidence can be obtained by comparing the groups which interact with the substrate to provide binding and catalysis with those which interact with the putative transition-state inhibitor. The Km value for inosine binding to IU-nucleoside hydrolase is independent of pH over the range 5.6-10.5. Dependencies of Vmax and Vmax/Km on pH result in pH optima near 8.0. A single group with pK of 9.1 must be protonated for catalytic activity, and protonation of a second group with a pK of 7.1 results in loss of activity. 1-(S)-Phenyl-1,4-dideoxy-1,4-imino-D-ribitol (phenyliminoribitol) binds with an equilibrium Kd of 30 nM and has been proposed to be a transition-state inhibitor. The pH dependence for the competitive inhibition by phenyliminoribitol resembles the Vmax profile with the protonation of a single group, pK 7.5, required for inhibitor binding and the protonation of a subsequent group, pK 6.6, causing loss of binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator.

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D. W., & Schramm, V. L. (1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by "arying the transition-state structure and comparing changes in the calculated kinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Vmax approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to C1' in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increase the catalytic rate.

What do international comparisons of health care expenditures really show?

There is much interest in international comparisons of health care expenditures, in particular their relation to national income. They have been widely used to judge countries' performance in cost-containment, and in the United Kingdom have been widely quoted in debates about the funding of the National Health Service. This paper challenges conclusions drawn from simple analyses of this topic, which have used dubious and inappropriate data, questionable methods and assumptions, and simplistic ad-hoc reasoning. It looks particularly at price differences between countries, which have usually been hidden by using exchange rates to standardize national figures. When more appropriate conversion factors called purchasing power parities are used, many of the simple and conventionally-accepted conclusions no longer appear so obvious. The attempt to create apparent scientific facts for policy debates has been based on a misuse of international comparisons.

Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei. Sequence, expression, and molecular analysis.

N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size-polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in blood-stream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21 (DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to > 97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine.

Adenosine 5'-phosphate was synthesized with 3H or 14C label specifically located as [1'-3H]AMP, [1'-14C] AMP, [5'-3H]AMP, and [5'-14C]AMP. The synthesis was accomplished from adenine and glucose or adenine and ribose using enzymes from the pentose pathway and/or from the purine salvage pathways. Structural analysis of the compounds confirmed the locations of the radiolabels. The methods provide a general scheme for the efficient synthesis of adenine nucleotides of high purity with 3H or 14C at any stable position on the ribose ring. Synthesis of [5'-14C]dAMP and [1'-3H] dAMP from the corresponding ribonucleotides was accomplished with ribonucleotide reductase. Labeled inosine was prepared by enzymatic dephosphorylation and deamination of labeled AMP. These compounds have been used to measure the secondary kinetic isotope effects on the acid-catalyzed hydrolysis of the N-glycosidic bond of AMP, dAMP, and inosine and the corresponding primary kinetic isotope effects with AMP. Acid hydrolysis in 0.1 or 0.2 N HCl at 50 degrees C gave 1H/3H secondary kinetic isotope effects of 1.23 +/- 0.01, 1.26 +/- 0.01, and 1.230 +/- 0.003 for AMP, dAMP, and inosine, respectively. The primary kinetic isotope effect for 12C/14C was 1.049 +/- 0.010 for AMP. The apparent rate constants for hydrolysis under these conditions were similar for inosine and AMP and were in the range 10(-6)-10(-5)s-1. Acid hydrolysis of dAMP is approximately 1000-fold faster than AMP but gives a similar 1H/3H kinetic isotope effect. The results of secondary isotope effects indicate that the transition states for the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to 1'-3H in the transition state and have considerable carboxonium character. Results with [1'-14C]AMP demonstrate that a significant primary isotope effect can be measured in the acid solvolysis of the N-glycosidic bond of AMP.

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction.

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.

Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata.

The transition state of nucleoside hydrolase from the trypanosome Crithidia fasciculata has been characterized by multiple Vmax/Km kinetic isotope effects with labeled inosine and adenosine as substrates. Nucleoside hydrolase catalyzes the hydrolysis of the N-glycosidic linkage of the commonly occurring purine and pyrimidine nucleosides, with Vmax/Km ranging over 2 orders of magnitude. The kinetic isotope effects for inosine were [1'-3H] = 1.150 +/- 0.006, [2'-3H] = 1.161 +/- 0.003, [1'-14C] = 1.044 +/- 0.004, [9-15N] = 1.026 +/- 0.004, [4'-3H] = 0.992 +/- 0.003, and [5'-3H] = 1.051 +/- 0.003. The magnitude of the kinetic isotope effects for inosine, an equivalent [1'-3H] kinetic isotope effect for the poor substrate adenosine, and the rapid equilibrium random kinetic mechanism [Parkin D, W., Horenstein, B. A., Abdulah, D. R., Estupiñán, B., & Schramm, V. L. (1991) J. Biol. Chem. (in press)] all indicate that the isotope effects are fully expressed. The kinetic and solvent deuterium isotope effects have been used to analyze the transition-state structure using bond energy bond order vibrational analysis. The transition state involves a protonated hypoxanthine leaving group with a C-N glycosidic bond elongated to approximately 2 A. The ribose group contains substantial carbocationic character, unusually strong hyperconjugation of H2', and a bond length of approximately 3 A to the incoming oxygen nucleophile. The remote isotope effect (4'-3H and 5'-3H) and the results of transition-state calculations provide the most detailed description of the steric and bonding properties of an enzyme-stabilized transition state.

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.