Effects of thermodynamic nonideality on the kinetics of ester hydrolysis by alpha-chymotrypsin: a model system with preexistence of the isomerization equilibrium

The effects of a small inert solute, sucrose, on the kinetics of hydrolysis of N-acetyl-tryptophan ethyl ester by bovine alpha-chymotrypsin have been investigated. In studies at pH 7 and 20 degrees C the presence of 0.5 M sucrose in assay mixtures caused no discernible change in kinetic parameters, a result consistent with existence of the enzyme in a single conformational state under those conditions. However, at pH 3.5 and 50 degrees C, conditions under which the enzyme comprises an equilibrium mixture of compact and expanded isomeric states, inclusion of the inert solute led to a considerable decrease in Michaelis constant (0.84 to 0.61 mM) but no significant change in maximal velocity. These results were shown to be amenable to quantitative interpretation in terms of thermodynamic nonideality effects on catalysis by an enzyme undergoing reversible isomerization in the absence of substrate. For that analysis, which required experimental estimates of the equilibrium constant for preexisting isomerization of enzyme and the activity coefficient of substrate, the magnitude of the former (0.3) was obtained by difference spectroscopy: liquid-liquid partition studies with bromobenzene as organic phase were used to determine the effect of sucrose on the activity coefficient of N-acetyltryptophan ethyl ester. Such agreement between experimental kinetic findings and theoretical predictions based on considerations of excluded volume points to the possible use of the space-filling effects of small solutes for delineating the gross extent of conformational changes associated with reversible isomerization of proteins, and hence to the potential of thermodynamic nonideality as a probe for studying protein denaturation mechanisms as well as substrate-mediated changes associated with enzyme reaction mechanisms.     

Recognition of Ribose Moiety by Adenosine (Phosphate) Deaminase from Squid Liver

An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5′-deoxyadenosine than that for adenosine, 2′-, or 3′-deoxyadenosine. 3′-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3′ phenylphosphonate by the enzyme, but 5′-AMP and 5′-IMP did not. The enzyme deaminated the 5′-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3′-hydroxyl terminal one in dinucleotides. The 5′-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.     

Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: invertase-catalyzed hydrolysis of sucrose

Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived. Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 degrees C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state. Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system [J. M. Nelson and M. P. Schubert (1928) J. Amer. Chem. Soc. 50, 2188-2193] also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Activity of adenosine deaminase and adenylate deaminase on adenosine and 2', 3(')-isopropylidene adenosine: role of the protecting group at different pH values

The deamination rate of 2',3'-isopropylidene adenosine catalyzed by adenosine deaminase (ADA) from calf intestine and adenylate deaminase (AMPDA) from Aspergillus species has been evaluated and compared with that of the enzymatic reactions of adenosine, to elucidate the influence of the protecting group on enzyme activity.     

Isolation of mutant adenosine deaminase by coformycin affinity chromatography

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.     

Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization

The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s?1 at 30 °C. Since adenine is deaminated ～103 slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.     

Deamination of adenosine monophosphate in the rat brain in hyperoxia, hypoxia, and cold stress]

The causes of the adenosine monophosphate (AMP) deamination increase in rat brain mitochondria under conditions of hyperoxia, hypoxia and cold stress were studied. Data from the inhibitory analysis suggest that the increased intensity of AMP deamination under hypoxia is conditioned by the alterations in the substrate specificity of type A monoamine oxidase which acquires the ability to deaminate AMP. The enhancement of AMP deamination under hyperoxia and cold stress is due to the activation of true AMP deaminase in the mitochondrial fraction. The cytoplasmic AMP deaminase activity remains unchanged thereby. The effects of the AMP deaminase specific effectors, ATP and inorganic phosphate, were investigated.     

Induction of Adenosine Deaminase in Escherichia coli

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.     

Aspects of purine metabolism in the gill epithelium of rainbow trout, Salmo gairdneri Richardson.

1. Enzymes interconnecting the adenylate pool were present in high concentration. 2. AMP and adenosine were easily deaminated by the corresponding enzymes whose high levels were detected. 3. Adenylate was hydrolyzed either by deamination to yield IMP which was further dephosphorylated to inosine or by dephosphorylation to adenosine followed by deamination to inosine. 4. Incubation of gill extract with [-14C]-AMP in the presence and absence of ATP but with adenosine deaminase inhibitors allowed demonstration that ATP controlled the balance between these pathways. 5. Some biochemical properties of 5'-nucleotidase. AMP deaminase and adenosine deaminase were defined. 6. Purine salvage enzymes were also estimated.     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products.     

Folic acid and pterin deaminases in Dictyostelium discoideum: kinetic properties and regulation by folic acid, pterin, and adenosine 3'',5''-phosphate.

Kinetic data obtained for deamination of pterin by the extracellular fraction from Dictyostelium discoideum yielded apparently linear Lineweaver-Burk plots for pterin. The Michaelis constant for pterin was 30 microM. The data for folic acid deamination yielded convex Lineweaver-Burk plots. Convex Lineweaver-Burk plots could result from the presence of two types of enzymes with different affinities. The data for folic acid deamination were analyzed mathematically for two types of enzymes. This analysis produced Michaelis constants for folic acid of 1.8 and 23 microM competition studies suggested that an enzyme with low affinity nonspecifically catalyzed the deamination of folic acid and pterin, whereas an enzyme with high affinity was a specific folic acid deaminase. A specific folic acid deaminase with high affinity appeared to be present on the surface of D. discoideum cells. The Michaelis constant for this enzyme was 2.6 microM. Cells growing in nutrient broth and cells starved in phosphate buffer released folic acid and pterin deaminases. The quantity of deaminase activities released by the cells appeared to be controlled by chemoattractants. Starving cells that were supplied with folic acid, pterin, or adenosine 3',5'-phosphate increased their extracellular folic acid and pterin deaminase activities to a larger extent than did cell suspensions to which no chemoattractants were added. Administration of folic acid or pterin to starving cells caused increases of the activity of extracellular adenosine 3',5'-phosphate phosphodiesterase and repressed increases of the activity of phosphodiesterase inhibitor.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Influence of stabilizers cosolutes on catalase conformation

The effects of sucrose, mannitol and betaine on the thermodynamic stability and the conformational state of the catalase enzyme were analyzed in order to understand the molecular mechanism whereby the solutes stabilized the enzyme. Catalase was selected as the model enzyme because it is used in several biotechnological processes. In the presence of each cosolute, our data have shown that there was a significant increase in the thermal stability of catalase. A minor stabilization in the enzyme secondary structure were induced by these cosolutes, as circular dichroism in the far UV region has demonstrated. Furthermore, our results support the idea that the overall native structure of catalase becomes more rigid, at least in certain surface areas, in the presence of the assayed stabilizers. This last finding can be reasonably explained by the exclusion mechanism of cosolutes from the protein surface which increases the structured water around this area.     

Complete release of adenosine deaminase from mouse lymphocytes stabilized by low-pH acetate

Complete release of adenosine deaminase from mouse lymphocytes takes place when intact cells are stabilized by low-pH acetate buffer. Both the low pH and the acetate affect the enzyme extraction markedly. At pH 5.0 all the adenosine deaminase activity detectable in the whole cell homogenates is released into the acetate buffer in very few minutes, with a total amount of 2% protein being extracted. The complete extraction of the enzyme activity is never observed when, at pH 5.0, the acetate is replaced by glutamate, citrate, succinate or maleate and only 45% and 15% of the adenosine deaminase activity is extracted by the acetate at pH 6.0 and 7.0, respectively. The breakdown of adenosine by the enzyme activity extracted from the stabilized cells is due to deamination alone, since inosine is the only product of the catalyzed reaction and its formation is completely inhibited by coformycin, a selective inhibitor of adenosine deaminase. The enzyme extracted shows a specific activity 50-times higher than that found in the crude homogenates, and a substantial purification of the enzyme extracted is achieved by a single Sephadex G-100 gel filtration.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.     

Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase

The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-)(1) and K(M) = 0.83 +/- 0.22 microM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 microM). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.