Studies on inosine monophosphate dehydrogenase. Isotope exchange at equilibrium.

Investigations on the mechanism of the IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14) reactions have been made at pH 7.0 by measuring rates of isotope exchange at chemical equilibrium with K+ maintained at a constant concentration. The results are generally in accord with the conclusions reached on the basis of the steady-state kinetic data obtained previously and confirm that there is random addition of IMP and NAD to the enzyme. The data also indicate clearly that at pH 7.0 catalysis is faster than the rate of IMP and/or XMP release which is rate limiting for the reaction sequence. The binding of IMP to the enzyme at pH 8.1 has been demonstrated to occur in the absence of both K+ and NAD and id independent of the K+ concentration.     

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.     

Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes.

Steady-state kinetic techniques have been used to investigate each of the reactions catalyzed by the bifunctional enzyme, chorismate mutase-prephenate dehydrogenase, from Aerobacter aerogenes. The results of steady-state velocity studies in the absence of products, as well as product and dead-end inhibition studies, suggest that the prephenate dehydrogenase reaction conforms to a rapid equilibrium random mechanism which involes the formation of two dead-end complexes, viz, enzyme-NADH-prephenate and enzyme-NAD+-hydroxyphenylpyruvate. Chorismate functions as an activator of the dehydrogenase while both prephenate and hydroxyphenylpyruvate acted as competitive inhibitors in the mutase reaction. By contrast. bpth NAD+ and NADH function as activators of the mutase. Values of the kinetic parameters associated with the mutase and dehydrogenase reactions have been determined and the results discussed in terms of possible relationships between the catalytic sites for the two reactions. The data appear to be consistent with the enzyme having either a single site at which both reactions occur or two separate sites which possess similar kinetic properties.     

Human liver akdehyde dehydrogenase. Kinetics of aldehyde oxidation.

Steady state initial velocity studies were carried out to determine the kinetic mechanism of human liver aldehyde dehydrogenase. Intersecting double reciprocal plots obtained in the absence of inhibitors demonstrated that the dehydrogenase reaction proceeded by sequential addition of both substrates prior to release of products. Dead end inhibition patterns obtained with coenzyme and substrate analogues (e.g. thionicotinamide-AD+ and chloral hydrate) indicated that NAD+ and aldehyde can bind in random fashion. The patterns of inhibition by the product NADH and of substrate inhibition by glyceraldehyde were also consistent with this mechanism. However, comparisons between kinetic constants associated with the dehydrogenase and esterase activities of this enzyme suggested that most of the dehydrogenase reaction flux proceeds via formation of an initial binary NAD+-enzyme complex over a wide range of substrate and coenzyme concentrations.     

The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.     

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.     

Investigations on the kinetic mechanism of octopine dehydrogenase. 1. Steady-state kinetics.

The kinetic mechanism of action of octopine dehydrogenase was investigated. This enzyme catalyses the reversible dehydrogenation of D-octopine to L-arginine and pyruvate, in the presence of nicotinamide-adenine dinucleotide. Initial velocity and product inhibition studies were carried out in both directions. Most of the results are consistent with a bi-ter sequential mechanism where NAD+ binds first to the enzyme followed by D-octopine, and the products are released in the order L-arginine, pyruvate and NADH. Various kinetic parameters were determined for each reactant at 33 degrees C, at pH 9.6 for NAD reduction, at pH 6.6 for NADH oxidation.     

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)     

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.     

Octopine dehydrogenase from Pecten maximus: steady-state mechanism

The steady-state kinetic mechanism of the reaction catalyzed by octopine dehydrogenase [N2-(1-carboxyethyl)-L-arginine:NAD+ oxidoreductase] was investigated at pH 6.9 and 9.2 by studies of substrate inhibition, analogue inhibition, and product inhibition. In the direction of octopine synthesis, the inhibition patterns in the presence of delta- guanidinovalerate and propionate show that NADH binds to the enzyme first followed by L-arginine and pyruvate which bind randomly. In the direction of octopine oxidation, the substrate patterns show that NAD binds to the enzyme before octopine in a rapid equilibrium fashion, and the product inhibition patterns show that the products L-arginine and pyruvate are released in a random fashion. Double, synergistic, substrate inhibition by L-arginine and pyruvate was shown to be due to binding (hypothetically of the imine) to the free enzyme and the enzyme-NAD complex. Furthermore, an alternate minor pathway was demonstrated which includes an enzyme-NADH-octopine complex and an enzyme-octopine complex.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii

Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.     

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.     

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.     

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)     

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.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.