Abortive Complex Formation Research Articles

A mechanism for bistability in glycosylation.

Glycosyltransferases are a class of enzymes that catalyse the posttranslational modification of proteins to produce a large number of glycoconjugate acceptors from a limited number of nucleotide-sugar donors. The products of one glycosyltransferase can be the substrates of several other enzymes, causing a combinatorial explosion in the number of possible glycan products. The kinetic behaviour of systems where multiple acceptor substrates compete for a single enzyme is presented, and the case in which high concentrations of an acceptor substrate are inhibitory as a result of abortive complex formation, is shown to result in non-Michaelian kinetics that can lead to bistability in an open system. A kinetic mechanism is proposed that is consistent with the available experimental evidence and provides a possible explanation for conflicting observations on the β-1,4-galactosyltransferases. Abrupt switching between steady states in networks of glycosyltransferase-catalysed reactions may account for the observed changes in glycosyl-epitopes in cancer cells.

Real-Time Single-Molecule Tethered Particle Motion Experiments Reveal the Requirements for Catalysis in the Tyrosine-Family Recombinase Active Site

Flp recombinase, a member of tyrosine family site-specific recombinases, is important to maintain the copy number in Saccharomyces cerevisiae via the DNA rearrangements, including insertions, deletions and inversions. It has been known that its catalytic activity is regulated by the complicated conformation change in the the protein-protein contact. Even though detailed structural analysis and kinetic measurements have been done, more evidences indicated there is significant difference among tyrosine family recombinases, Cre and Flp. Here, the reaction process catalyzed by Flpe, a variant of Flp known to be more active at 37 °C than wild type, was investigated by single-molecule tethered particle motion (TPM) from start to the end. There are early formation of abortive complexes and the presence of wayward complexes in the Flpe-FRT system, similar to the Δ Int and Cre recombinases. Moreover, the presence of a Holliday junction intermediate served as a rate-limited barrier was found. Different from the Cre recombinase, the kinetic analyses on the synapse state duration indicated the irreversibility in the strand cleavage/ligation process. The new information offered here demonstrates that single-molecule approaches can be the potential methodologies to explore reaction mechanisms in different perspectives.

Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

Yeast sterol C24-methyltransferase: Role of highly conserved tyrosine-81 in catalytic competence studied by site-directed mutagenesis and thermodynamic analysis

The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction ( k cat/ K m) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of E a, Δ G ‡, Δ H ‡ and TΔ S ‡) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24( R,S)25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as K m/ K i), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation.

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6xHis-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.

The Contribution of a Conformationally Mobile, Active Site Loop to the Reaction Catalyzed by Glutamate Semialdehyde Aminomutase

The behavior of glutamate semialdehyde aminomutase, the enzyme that produces 4-aminolevulinate for tetrapyrrole synthesis in plants and bacteria, is markedly affected by the extent to which the central intermediate in the reaction, 4,5-diaminovalerate, is allowed to dissociate. The kinetic properties of the wild-type enzyme are compared with those of a mutant form in which a flexible loop, that reversibly plugs the entrance to the active site, has been deleted by site-directed mutagenesis. The deletion has three effects. The dissociation constant for diaminovalerate is increased approximately 100-fold. The catalytic efficiency of the enzyme, measured as k(cat)/K(m) in the presence of saturating concentrations of diaminovalerate, is lowered 30-fold to 2.1 mM(-1) s(-1). During the course of the reaction, which begins with the enzyme in its pyridoxamine form, the mutant enzyme undergoes absorbance changes not seen with the wild-type enzyme under the same conditions. These are proposed to be due to abortive complex formation between the pyridoxal form of the enzyme (formed by dissociation of diaminovalerate) and glutamate semialdehyde itself.

Purine nucleoside phosphorylase from bovine lens: purification and properties

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1.) was purified 38 750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97 000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15 250 and 14 650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

A kinetic study of rat recombinant DNA polymerase beta: detection of a slow (hysteretic) transition in polymerase activity and inhibition by butylphenyl-deoxyguanosine triphosphate.

We have identified and characterized a distinct non-linearity in the time course of the reaction of mammalian DNA polymerase beta with synthetic polynucleotides. Nucleotide incorporation is biphasic; an initial burst of activity decays exponentially to a lower steady-state velocity. This slow transition in polymerase activity is not due to substrate depletion, abortive complex formation, or enzyme inactivation. The data are consistent with description of the beta-polymerase as a hysteretic enzyme, a finding which provides a potential explanation for the non-hyperbolic kinetics which have been reported previously for this polymerase. We have also found, in contrast to some previous data, that the nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPdGTP), is an inhibitor of the beta-polymerase. When poly(dC).oligo(dG) is used as template.primer, inhibition of the initial velocity is competitive with dGTP with a Ki of 1.25 microM. On activated DNA, however, beta-polymerase displays sensitivity to BuPdGTP which overlaps with that previously reported for DNA polymerase delta; 100 microM BuPdGTP is required to inhibit the initial velocity of a dGTP-deficient, truncated assay. Finally, we demonstrate that, in addition to its inhibition of initial velocity, BuPdGTP also modulates both the rate constant of the slow transition in polymerase activity, and the steady-state velocity of the beta-polymerase.

Kinetic properties and characteristics of mouse liver mitochondrial asparagine aminotransferase

Mouse liver asparagine aminotransferase has been found to be a mixture of enzyme forms having a cytosolic component and a mitochondrial component. The molecular weight of the mitochondrial enzyme is 70,800. The mitochondrial asparagine amino-transferase is strongly inhibited by aminooxyacetate. It is less affected by d-cycloserine but a small amount of inhibition is observed. Cysteine strongly inhibits the enzyme as do several sulfhydryl modifying reagents. The activities of the cytosolic and mitochondrial aminotransferases have been separated, and the kinetic properties of the mitochondrial form determined. The mouse liver mitochondrial asparagine aminotransferase is fairly specific for asparagine, utilizing very few amino acids as alternate amino donors and none to a great extent. The keto acid specificity is very broad, but glyoxylate is one of the most active amino group acceptors. The kinetic properties of the mitochondrial enzyme are also reported here and the data indicate strong substrate and product inhibition. Abortive complex formation may account for the deviation of the double reciprocal plots from the expected pattern.

Kinetic characterization of the inhibition of purified cynomolgus monkey lactate dehydrogenase isozymes by gossypol.

This report describes the results of the first step in a sequence of experiments designed to test the hypothesis that the sperm-specific isozyme of lactate dehydrogenase (LDH-C4), is a site of action of the potential male contraceptive agent gossypol. Cynomolgus monkey LDH-A4, LDH-B4 and LDH-C4 were purified and kinetically characterized. LDH-A4 and LDH-B4 exhibited "linear mixed-type" inhibition by gossypol with both lactate and pyruvate as variable substrates. LDH-C4 also exhibited "linear mixed-type" inhibition with lactate as substrate. However, the C4 isozyme exhibited "parabolic mixed-type" inhibition by gossypol and substrate inhibition with pyruvate as substrate, the latter due to abortive complex formation. Of the three isozymes, LDH-C4 exhibited the lowest apparent Km for pyruvate and the highest apparent Km for lactate. The LDH-C4 form was found to be the most sensitive isozyme to gossypol inhibition, since it had the lowest apparent Ki values for gossypol inhibition. The effect of gossypol on coenzyme binding to LDH-C4 was examined and gossypol binding was found to inhibit binding and release of NADH but not NAD+, an effect possibly due to its interaction with the more hydrophobic loop region of LDH-C4.

Mechanistic origin of the kinetic cooperativity of hexokinase type L1 from wheat germ.

A theoretical analysis is presented which shows that initial velocity data for hexokinase L1 catalysis of glucose phosphorylation by MgATP cannot be reconciled with the observed rate of the 'mnemonical' conformational transition which has been proposed to account for the kinetic cooperativity of the enzyme. The basic kinetic properties of hexokinase L1 and other allegedly 'mnemonical' enzymes appear to be fully consistent with an ordered ternary-complex mechanism in which the leading substrate participates in abortive-complex formation. It is concluded that, so far, no enzyme displaying kinetic cooperativity has been convincingly demonstrated to operate by a 'mnemonical' type of reaction mechanism.

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

Equilibrium Kinetic Studies of Enzyme Mechanism and Control

This chapter discusses equilibrium kinetic studies of enzyme mechanism and control. It focuses on some aspects of the isotopic exchange kinetics at equilibrium and its evolution from the study of enzymic catalysis by the manipulation of reactants to the study of the control of catalysis by ligands that are not, themselves, catalytically transformed, inhibitors and activators, particularly of allosteric enzymes. The chapter presents particular areas of usefulness of these measurements with some examples. An important theroretical use of isotopic exchange at equilibrium was suggested to be the elucidation of compulsory pathways or binding orders. The usefulness of isotopic exchange kinetics for elucidation of compulsory binding orders was first established for the bovine heart and rabbit muscle lactate dehydrogenases. These enzymes were believed to have a compulsory pathway on the basis of binding and kinetic data. Subsequently, a compulsory pathway was demonstrated for porcine and bovine heart malate dehydrogenases and bovine liver glutamate dehydrogenase at pH 8.8. The study of enzyme catalysis at equilibrium by isotopic exchange of reactants is useful for elucidation of aspects of enzyme mechanism which may not be readily obtainable by other means. These include elucidation of a compulsory binding order, indications of fast and slow dissociation steps and whether chemical transformation is rate limiting; determination of the possibility of abortive complex formation, and, importantly, the observation of non-rate-limiting steps in catalysis. Another important aspect of equilibrium kinetics is its extension to the study of the mechanism of modifiers of enzymic catalysis, particularly with respect to effectors of allosteric enzymes.

The interactions of hydrazine derivatives with rat-hepatic cytochrome P-450.

The ability of different classes of hydrazine derivatives to modify cytochrome P-450 function during turnover as judged by loss of absorbance at 416 nm, loss of CO-reactive cytochrome P-450, or destruction of haem has been studied. Addition of monosubstituted hydrazines to rat-liver microsomes caused considerable loss of CO-reactive cytochrome P-450 and haem destruction; monosubstituted hydrazides caused mainly loss of CO-reactive cytochrome P-450, most likely due to abortive complex formation. Metabolism of 1,1-disubstituted hydrazines by microsomal cytochrome P-450 resulted in loss of CO-reactive cytochrome P-450 only, with no haem destruction. The 1,2-disubstituted hydrazines and hydrazides, procarbazine and iproniazid, acted similarly to the monosubstituted hydrazines, while 1,2-dimethylhydrazine elicited no response, either in observable spectral changes or loss of CO-reactive cytochrome P-450. Synthetic diazene intermediates of phenylhydrazine and N-aminopiperidine reacted rapidly with microsomal cytochrome P-450 to form a spectral intermediate resembling the putative iron porphyrin-diazenyl complex. The decomposition of certain iron porphyrin-diazenyl derivatives apparently leads to destruction of the porphyrin prosthetic group, most likely due to haem alkylation.

The effect of cryosolvents on the spectral and catalytic properties of liver alcohol dehydrogenase.

The effects of aqueous organic cryosolvents on the structural and catalytic properties of horse liver alcohol dehydrogenase (liver alcohol dehydrogenase) have been investigated. The cosolvents studied were ethanol, methanol, dimethyl sulfoxide, and dimethylformamide. All show potential as cosolvents for cryoenzymological investigations of the catalytic action of liver alcohol dehydrogenase. Limitations due to the formation of abortive complexes, or the cosolvent acting as a substrate were considered. Possible adverse structural effects of the cosolvents were ascertained by utilizing the intrinsic fluorescent properties of the enzyme. Catalytic effects, as inferred from steady state kinetic studies, were determined from both the oxidation of ethanol and the reduction of p-nitroso-N,N-dimethylaniline, a chromophoric aldehyde analog. It is concluded that each of these solvent systems may be useful for studying certain aspects of the liver alcohol dehydrogenase catalytic mechanism at subzero temperatures. Thus, although the formation of ternary enzyme-cosolvent complexes may restrict the use of cosolvents in some experiments, no apparent adverse effects are observed on the enzyme structure, coenzyme binding, or catalytic reactions. A number of interesting features were observed. For example, fluorescence titration of the native enzyme near 0 degrees C in either aqueous solution or 50% dimethyl sulfoxide revealed pK values in the vicinity of 10.5 and 12.5, in contrast to the previously reported single pK of 9.2 observed in aqueous solution at 25 degrees C.

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[ a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.

Aurintriboxylic acid as both a stimulator and an inhibitor of cell-free protein synthesis by rat heart polyribosomes

Aurintricarboxylic acid is shown to be a powerful stimulator at lower concentrations and a strong inhibitor at higher concentrations of protein synthesis in a heart ribosomes brain supernatant cell free system. The stimulation appears to arise from the prevention of low affinity abortive complex formation.

16] Product inhibition and abortive complex formation

16] Product inhibition and abortive complex formation

Constant-ratio alternative substrate approach for differentiation of enzyme mechanisms

A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available.

Evidence of a novel role for monovalent cations in pyruvate kinase catalysis.

In the pyruvate kinase (EC 2.7.1.40) reaction, the complete enzyme-products complex consists of enzyme, pyruvate, ATP, a divalent cation, and a monovalent cation, usually K+. The dissociation of this complex can be slow permitting reversible enolization of pyruvate in this complex during the course of the forward reaction. High concentrations of each component in the enzyme-products complex inhibits the forward reaction, but only elevated concentrations of the monovalent cation decrease the net rate of product dissociation relative to that of pyruvate enolization. This result indicates that the monovalent cation can be the first component released from the enzyme-products complex and that the presence of an inhibiting monovalent cation concentration leads to re-formation of the complete complex, which is necessary for pyruvate enolization. The evidence suggests that the monovalent cation can bind and release with each turnover of the enzyme. While the data do not permit the conclusion that first release of monovalent cation is the exclusive pathway for dissociation of the enzyme-products complex, no other component once released can reassociate rapidly enough to form a complete complex during the forward reaction. Inhibition by these components must be attributed to the formation of abortive complexes.