Ternary Complex Mechanism Research Articles

Divalent ion effects and insights into the catalytic mechanism of protein tyrosine kinase Csk.

Csk (C-terminal Src kinase) is a protein tyrosine kinase which catalyzes the transfer of the gamma-phosphoryl group of ATP to the tyrosine hydroxyl of proteins in the presence of a divalent ion. Previous work with poly(Glu,Tyr) as the tyrosine-containing substrate and Mn as the divalent ion defined a ternary complex mechanism with ADP product release partially rate-determining [Cole, P. A., et al. (1994) J. Biol, Chem. 269, 30880-30887]. In this current study, ionic strength and divalent ion effects were probed. Increasing ionic strength led to a dramatic rise in the poly(Glu,Tyr) [4:l poly(glutamate:tyrosine)] K(m) and had little effect on the ATP K(m) or Kcat in Csk-mediated phosphoryl transfer. This finding allowed the dead-end peptide inhibitor EDNEFTA to be characterized as a linear competitive inhibitor of poly(Glu,Tyr) and a linear noncompetitive inhibitor of ATP. Taken together with previous data, the overall kinetic mechanism could now be assigned as random substrate binding, ternary complex. Compared to Mn, Mg was shown to sustain phosphoryl transfer with a 2.5-fold higher Kcat but K(m)'s for ATP and poly(Glu,Tyr) that were some 15-20-fold higher. An elevated ADP Ki and microviscosity effects were most suggestive of a kinetic mechanism with fast ADP release, and the chemical step fully rate-determining in the Mg-dependent reaction. Steady-state kinetic analyses of Csk reactions with Co and Ni in addition to Mg and Mn on wild-type and D314E Csk with ATP and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)] as substrates were performed. The Kcat thio effects [Kcat(ATP)/Kcat(ATP gamma S)] were inversely correlated with metal thiophilicity in both wild-type and D314E mutant Csk reactions, although the relationship was less pronounced in the latter. These results appear to underscore the role of gamma-phosphoryl hydrogen bonding/salt bridging in the wild-type Csk reaction transition state, which is somewhat perturbed in the D314E Csk reaction. In the case of the Ni reaction, the Kcat thio effect was reduced to about 2 in the wild-type and D314E mutant Csk reactions. Relevance with regard to the degree of nucleophilic attack in the transition state, i.e., associative vs dissociative character of phosphoryl transfer, is discussed.

Enzyme-monitored turnover of Escherichia coli thioredoxin reductase: insights for catalysis.

Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit. It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis. Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound. Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed. It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme. When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound. These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis.

Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination.

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates. A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C. Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction. Additional, faster routes for product release are achieved via the formation of the nonproductive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm). The release of 3-acetyl-Cm from the binary complex is 5-fold slower than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively. Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range. Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model. Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the kinetic mechanism of enterococcal NADH peroxidase reveals catalytic roles for NADH complexes with both oxidized and two-electron-reduced enzyme forms.

Anaerobic titrations of the two-electron-reduced NADH peroxidase (EH2) with NADH and 3-acetylpyridine adenine dinucleotide (AcPyADH) yield the respective complexes without significant formation of the four-electron-reduced enzyme (EH4). Further analysis of the EH2/EH4 redox couple, however, yields a midpoint potential of -312 mV for the free enzyme at pH 7. The catalytic mechanism of the peroxidase has been evaluated with a combination of kinetic and spectroscopic approaches, including initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzyme (E) and EH2 with NADH and AcPyADH, and diode-array spectral analyses of both the reduction of E-->EH2 by NADH and the formation of EH2.NADH. Overall, these results are consistent with rapid formation of an E.NADH complex with distinct spectral properties and a rate-limiting hydride transfer step that yields EH2, with no direct evidence for intermediate FADH2 formation. The EH2.NADH complex described previously [Poole, L. B., & Claiborne, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slowly with H2O2. Stopped-flow analyses do, however, support the very rapid formation of an EH2.NADH* intermediate, with spectral properties that distinguish it from the static EH2.NADH form, and yield a first-order rate constant for the conversion between the two species that is smaller than kcat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mechanism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R.P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167].

Human acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation.

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

Effect of pyridine nucleotide on the oxidative half-reaction of Escherichia coli thioredoxin reductase.

The kinetics of the oxidative half-reaction between reduced thioredoxin reductase and oxidized thioredoxin measured in the presence and absence of pyridine nucleotide show a significant difference in the rates of the main phase of oxidation. When 1 equiv of NADPH is used to partially reduce the enzyme at pH 7.0 or 7.6, the observed rate of the catalytically competent phase of oxidation is essentially equal to kcat at that pH. This is about 50% of the rate of oxidation observed with enzyme fully reduced or partially reduced by the xanthine/xanthine oxidase system or by dithionite. Through the use of the nonreducible analog 3-aminopyridine adenine dinucleotide phosphate we have shown that this decrease in observed rate of oxidation is linked to the concentration of pyridine nucleotide present. This suggests that the complexation of pyridine nucleotides with reduced thioredoxin reductase is able to effect a change in the rate-limiting steps of the oxidation of the enzyme by thioredoxin. This is the case even when substoichiometric quantities of 3-aminopyridine adenine dinucleotide phosphate are present, which predicts that the binding to reduced enzyme is very tight. It is clear that the presence of 1 equiv of NADP+ is sufficient to cause the observed rate for the catalytically competent phase of oxidation to decrease to kcat. Thus, there is compelling evidence for a ternary complex mechanism for thioredoxin reductase.

Purification and characterization of coenzyme F390 synthetase from Methanobacterium thermoautotrophicum (strain delta H).

Coenzyme F390 synthetase catalyzes the formation of 8-hydroxyadenylylated-coenzyme F420 (coenzyme F390-A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 synthetase from Methanobacterium thermoautrophicum was purified from a cell-free extract and its catalytic properties were determined. The synthetase was purified 150-fold to a specific activity of 0.45 mumol.min-1.mg protein-1. The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co-elution during purification of GTP-dependent coenzyme F390 synthetase activity suggested that the synthetase is also capable of 8-hydroxyguanylylated-coenzyme F420 (coenzyme F390-G) formation. Initial-velocity measurements of the two-substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary-complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 microM and a Km for ATP of 1.7 mM. In contrast to the enzyme in the cell-free extract, the isolated enzyme was active under aerobic and anaerobic conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5-dihydro-coenzyme F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 microM) with respect to coenzyme F420. The latter findings may explain why the enzyme could only be detected in crude extracts that had been exposed to air, i.e. treatment with air causes the oxidation of reduced coenzyme F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.

Purification and Characterization of Coenzyme F390 Synthetase from Methanobacterium thermoautotrophicum (strain H)

Coenzyme F390 synthetase catalyzes the formation of 8‐hydroxyadenylylated‐coenzyme F420 (coenzyme F390‐A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 synthetase from Methanobacterium thermoautrophicum was purified from a cell‐free extract and its catalytic properties were determined. The synthetase was purified 150‐fold to a specific activity of 0.45 μmol · mim−1· mg protein−1. The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co‐elution during purification of GTP‐dependent coenzyme F390 synthetase activity suggested that the synthetase is also capable of 8‐hydroxyguanylylated‐coenzyme F420, (coenzyme F390‐G) formation.Initial‐velocity measurements of the two‐substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary‐complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 μM and a Km for ATP of 1.7 mM.In contrast to the enzyme in the cell‐free extract, the isolated enzyme was active under aerobic and anaerobic conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5‐dihydro‐coenzyme F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 μM) with respect to coenzyme F420. The latter findings may explain why the enzyme could only be detected in crude extracts that had been exposed to air, i.e. treatment with air causes the oxidation of reduced coenzyme F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.

Ligand binding on to maize (Zea mays) malate synthase: a structural study

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

Kinetic analysis of lactate dehydrogenase using integrated rate equations.

The reaction catalyzed by lactate dehydrogenase was analyzed under fully second-order conditions using integrated rate equations. A two-step regression analysis was utilized to fit twenty-one progress curves repeated in sextuplicate to the general mechanism second-order integrated rate equation with additional terms of substrate inhibition. The fitting error was less than one percent. The resulting kinetic constants support a ternary complex mechanism; in no case were constants supporting another mechanism predicted. The inhibition constant for oxamate was also determined.

Chemical and kinetic mechanism of the inositol monophosphatase reaction and its inhibition by Li+.

Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li+], Li+ inhibited the hydrolysis of racemic myo-inositol 1-phosphate, myo-inositol 4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent Ki values of 1.1, 0.11 and 1.52 mM, respectively. At Li+ concentrations higher than 4 mM, Li+ acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, Ki greater than 1.5 mM. The enzyme was unable to catalyze the transesterification of [14C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li+ binds after P-O bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of 18O from [18O] water into phosphate was shown to be completely dependent upon inositol. The Km for inositol for 18O exchange was 190 mM and in the presence of saturating phosphate, VEx was at least 60% of Vmax for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li+ exerts its action by binding to enzyme/product complexes. At low concentration, Li+ inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol 1-phosphate and 2'AMP as the substrate but not with inositol 4-phosphate. The combined results indicate that Mg2+ and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (Ki = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li+ and phosphate, increasing [Li+] caused the Ki for phosphate to decrease by a factor of (1 + [Li+]/KLi). The Ki for the first product off (inositol) was, however, unaltered by Li+. The results indicate that Li+ can bind to the species E.Ins.Pi and E.Pi, but not to enzyme/substrate complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and analysis of streptococcal NADH peroxidase expressed in Escherichia coli.

Using the T7 RNA polymerase expression system, a modified plasmid vector has been developed which gives reliable, high level expression in Escherichia coli of the gene encoding streptococcal NADH peroxidase. The recombinant enzyme has been purified to homogeneity using a revised protocol which yields over 35 mg of pure protein per liter of culture. Recombinant NADH peroxidase is fully active and exhibits spectroscopic and redox properties identical to those for the enzyme purified from Streptococcus faecalis 10C1. Reductive titrations and thiol analyses confirm the presence of the unusual cysteine-sulfenic acid (Cys-SOH) redox center identified previously. N-terminal sequence analysis, analytical gel filtration, and preliminary x-ray diffraction data all confirm the structural identity of the recombinant and S. faecalis enzymes. Steady-state kinetic analysis of the peroxidase, coupled with results from static titration experiments is consistent with a limiting type of ternary complex mechanism and allows the determination of many of the corresponding kinetic constants. In addition, preliminary 1H NMR spectra of the enzyme at millimolar concentrations show good dispersion in the amide region and indicate that the recombinant peroxidase is suitable for one-dimensional NMR work with labeled amino acids.

Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates.

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.

Purification and properties of 5,10-methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase, two coenzyme F 420-dependent enzymes, from Methanosarcina barkeri

5,10-Methylenetetrahydromethanopterin dehydrogenase and 5,10-methylenetetrahydromethanopterin reductase have been purified to homogeneity by a factor of 86 and 68, respectively, from methanol-grown Methanosarcina barkeri cells. The dehydrogenase was isolated as a hexamer of a single 35 kDa subunit, whereas the reductase was composed of four identical 38 kDa subunits. The purified oxygen-stable enzymes catalyzed the oxidation of 5,10-methylenetetrahydromethanopterin and methyltetrahydromethanopterin with V max values of 3000 and 200 μmol min −1 mg −1, respectively. The methanogenic electron carrier coenzyme F 420 was a specific electron acceptor for both enzymes. Steady state kinetics for the two enzymes were in agreement with ternary complex (sequential) mechanisms. Methylene reductase and methylene dehydrogenase are proposed to function in the methanol oxidation step to CO 2.

Kinetic mechanism of monoamine oxidase A

Steady-state kinetic data for monoamine oxidase A in crude extracts suggest an exclusively ping-pong mechanism, in contrast to those for monoamine oxidase B, which indicate alternate mechanisms involving either a binary or ternary complex. In this study, with use of purified monoamine oxidase A, steady-state data for the inhibition by D-amphetamine of the oxidation of primary amines indicate the possibility of a ternary complex mechanism for monoamine oxidase A also. Stopped-flow studies demonstrate that the rate of reoxidation of reduced enzyme is enhanced by substrates but not by the product, 1-methyl-4-phenylpyridinium. Thus, for the A enzyme, the ternary complex with substrate, but not product, is reoxidized at a faster rate than the free, reduced enzyme. For both the A and B forms of monoamine oxidase, the mechanism is determined by competition between alternate pathways on the basis of the relative rate constants and dissociation constants.

Purification and characterization of coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain ΔH

5,10-Methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain ΔH was purified to homogeneity with nearly complete recovery. The aerobically stable monofunctional enzyme catalyzed the reversible oxidation of 5,10-methylene-5,6,7,8-tetrahydromethanopterin to its 5,10-methenyl derivative. For the reaction a midpoint potential Eo′ = −362 mV was calculated at 60°C. The methanogenic electron carrier coenzyme F420 was strictly required as the co-substrate. The dehydrogenase (Mr 216000) was purified as an apparent hexamer of six identical 36 kDa subunits. Oxidation of 5,10-methylenetetrahydromethanopterin coupled to coenzyme F420 reduction catalyzed by the dehydrogenase with a turnover number of 2400 s−1 proceeded via a ternary complex mechanism. High concentrations of monovalent cations markedly stimulated the reaction.

Inositol dependent phosphate-oxygen ligand exchange catalysed by inositol monophosphatase.

Inositol monophosphatase is unable to catalyse the exchange of 18O-label from 18O-water into inorganic phosphate in the absence of inositol. The inositol mediated exchange reaction is Mg 2+ dependent, shows a similar pH dependence to that for the hydrolysis of myo-inositol 1-phosphate and is inhibited by lithium cation. These results suggest that the enzyme operates via a ternary complex mechanism unlike alkaline and acid phosphatase.

Pharmacological estimation of agonist affinity: detection of errors that may be caused by the operation of receptor isomerisation or ternary complex mechanisms

1. Recent theoretical studies have questioned the pharmacological estimation of agonist affinity. They showed that when receptor isomerisation or ternary complex mechanisms operate, the receptor inactivation method can substantially overestimate affinity, whereas methods for partial agonist analysis are more accurate. We previously suggested that the operation of such mechanisms and therefore the presence of errors could be detected by analysing the same partial agonist by the receptor inactivation and comparative methods. This paper describes the practical application of this test. 2. The ternary complex mechanism was simulated for a partial agonist under various conditions relating receptor (R) and transducer (T) concentrations, one of which also corresponds to the receptor isomerisation mechanism. The theoretical data so generated were then analysed by the inactivation and comparative methods to quantify the magnitude of error of affinity estimation that could occur. 3. This analysis showed that for a partial agonist with approximately 85% of the activity of a full agonist, the inactivation method could produce an affinity (pKA) estimate up to 0.7 log10 units higher than that produced by the comparative method. This difference would occur when the total receptor concentration ([R0]) is less than or equal to the total transducer concentration ([T0]). It also showed that the overestimation of affinity by the inactivation method was accompanied by drastic overestimation of Em, the maximal effect parameter. 4. The test was then exemplified using the muscarinic receptor system in the guinea-pig isolated left atrial preparation, where there is evidence that a ternary complex mechanism operates. The test agonist was pilocarpine, which produced on average 83% of the activity of the full agonist, carbachol. Pilocarpine was analysed in comparison with carbachol and by receptor inactivation in the same tissue resulting in small and statistically insignificant differences in Em (96.7% and 97.3% respectively) and pKA (5.03 and 4.95 respectively). 5. In conclusion, in this experimental system, there was no evidence for the errors in agonist affinity estimation predicted by theory. Although this conclusion only applies to this system and application of the test to others is necessary to establish the generality of the present results, further examination of the theoretical basis for the predicted errors is required.

Kinetic mechanisms of mitochondrial carriers catalysing exchange reactions

The single-binding site or ping-pong mechanism is widely accepted for exchange reactions, catalysed by mitochondrial carriers. However, when the most relevant approach to discriminate between mechanisms, i.e., kinetic study is used, the ping-pong mechanism is eliminated in favour of the sequential or ternary complex mechanism implying two binding sites simultaneously accessible to both internal and external substrates. This is the case for the oxoglutarate carrier, the aspartate/glutamate carrier and there are very strong presumptive evidences for the adenylic carrier.