Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.     

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 properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.

The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.     

IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid

Mycophenolic acid (MA) was demonstrated to be an effective inhibitor of the growth of the intracellular parasitic protozoan Eimeria tenella in tissue culture and guanine was shown to reverse this inhibition as expected for an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205). A high performance liquid chromatography study of the intracellular nucleotide pools labeled with [3H]hypoxanthine was carried out in host cells lacking hypoxanthine-guanine phosphoribosyltransferase, and the depletion of guanine nucleotides demonstrated that the intracellular parasite enzyme was being inhibited by the drug. Kinetic studies carried out on the enzyme derived from E. tenella oocysts demonstrated substrate inhibition by NAD and mycophenolic acid inhibition similar to that found for mammalian enzymes, but different from that for bacterial enzymes. The inhibition by mycophenolic acid was not time-dependent and was immediately reversed upon dilution. As found previously for other IMP dehydrogenases, an Ordered Bi-Bi mechanism prevails with IMP on first followed by NAD, NADH off first, and then XMP. The kinetic patterns are consistent with substrate inhibition at high concentrations of NAD due to the formation of an E X XMP X NAD complex. Uncompetitive inhibition by MA versus IMP, NAD, and K+ was found and this was interpreted as evidence for the formation of an E X XMP X MA complex. A speculative mechanism for the inhibition of the enzyme is offered which is consistent with the fact that E X XMP X MA readily forms, whereas E X IMP X MA does not.     

L-fucose metabolism in mammals. Purification of pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase by NAD+-Agarose affinity chromatography.

Pork liver has previously been reported to contain a soluble enzymatic pathway which converts L-fucose to 2-keto-3-deoxy-L-fuconate and D-arabinose to 2-keto-3-deoxy-D-arabonate. We now report the isolation from pork liver of a soluble NAD+-dependent dehydrogenase which acts on both 2-keto-3-deoxy-L-fuconate and 2-keto-3-deoxy-D-arabonate. This enzyme has been purified to homogeneity by a five-step procedure; the final step involved affinity chromatography on NAD+-agarose. A purification factor of about 3000-fold was achieved with a yield of over 20%. The enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 9.1 and 7.0 and on the basis of sedimentation equilibrium analysis with the ultracentrifuge. The molecular weight of the native enzyme is about 100,000 while disc gel electrophoresis in the presence of sodium dodecyl sulfate and thiol showed the presence of a polypeptide of molecular weight 26,800; these results suggest that the enzyme is a tetramer. The enzyme has an isoelectric point of 5.4. The enzyme is unstable in the dilute state and in the absence of thiol but can be kept for 2 years at -70 degrees at a protein concentration of 4 mg per ml and in the presence of 1 mM dithiothreitol.     

Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of 相似文献   

Effect of pH on coenzyme binding to liver alcohol dehydrogenase.

1. The transient-state kinetics of ligand-displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on-velocity and off-velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 10. 2. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogues to the enzyme . NAD+ complex. 3. Critical experiments have been performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.     

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Transient kinetics of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.     

The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase.

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.     

Mechanism of bovine liver S-adenosylhomocysteine hydrolase. Steady-state and pre-steady-state kinetic analysis

The kinetic mechanism of S-adenosylhomocysteine hydrolase was investigated by stopped-flow spectrofluorometry at pH 7.0 and 25 degrees C. Pre-steady-state kinetic steps were identified with chemical steps proposed for the mechanism of this enzyme (Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217-1226). The steady-state kinetic constants for the hydrolysis or synthesis of S-adenosylhomocysteine were in good agreement with those values calculated from the pre-steady-state rate constants. The equilibrium constant for dehydration of 3'-ketoadenosine to 3'-keto-4',5'-dehydroadenosine on the enzyme was 3. The analogous equilibrium constant for addition of L-homocysteine to S-3'-keto-4',5'-dehydroadenosylhomocysteine on the enzyme was 0.3. The elimination of H2O from adenosine in solution had an equilibrium constant of 1.4 (aH2O = 1). Thus, the equilibrium constants for these elimination reactions on the enzyme were probably not perturbed significantly from those in solution. The equilibrium constant for the reduction of enzyme-bound NAD+ by adenosine was 8, and the analogous constant for the reduction of the enzyme by S-adenosylhomocysteine was 4. The equilibrium constant for the reduction of NAD+ by a secondary alcohol in solution was 5 x 10(-5) at pH 7.0. Consequently, the reduction of enzyme-bound NAD+ by adenosine was 10(5)-fold more favorable than the reduction of free NAD+. The magnitude of the first-order rate constants for the interconversion of enzyme-bound intermediates varied over a relatively small range (3-80 s-1). Similarly, the magnitude of the equilibrium constants among enzyme-bound intermediates varied over a narrow range (0.3-10). These results were consistent with the overall reversibility of the reaction.     

Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus.

Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.     

The role of metal in liver alcohol dehydrogenase catalysis. Spectral and kinetic studies with cobalt-substituted enzyme.

The kinetic and spectral properties of native and totally cobalt-substituted liver alcohol dehydrogenase have been compared. Based on titrimetric determinations of enzyme active site concentration, the turnover number at pH 7.0 for cobalt enzyme was the same as for native enzyme. At pH 10, however, the turnover number was slower for cobalt-substituted enzyme, 3.14 s-1 as compared with 4.05 s-1 for native enzyme. A comparison between native and totally cobalt-substituted enzyme showed a blue-shifted enzyme-NADH double difference spectrum and a splitting and red-shifted enzyme-NAD+-pyrazole double difference spectrum in the near-ultraviolet. The 655-nm peak of the cobalt-substituted enzyme was perturbed by the formation of enzyme-NADH binary complex, enzyme-NAD+-trifloroethanol ternary complex, and enzyme-NAD+ binary complex formation. At pH 7.0, the only observable step in the reaction sequence with a significantly different rate constant for cobalt enzyme was the catalytic hydrogen-transferring step. The rate constant for this step is 92 s-1 for totally cobalt-substituted enzyme as compared with 138 s-1 for native liver alcohol dehydrogenase. The results of this study indicate that zinc is involved in catalysis alcohol and NADH.