Characterization of a phosphoenzyme intermediate in the reaction of phosphoglycolate phosphatase

When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).     

Anion and divalent cation activation of phosphoglycolate phosphatase from leaves

Phosphoglycolate (P-glycolate) phosphatase was purified 223-fold from spinach leaves by (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and Sephadex G-200 chromatography. The partially purified enzyme had a broad pH optimum between 5.6 and 8.0 and was specific for the hydrolysis of P-glycolate with a Km (P-glycolate) of 26 microM. The enzyme was activated by divalent cations including Mg2+, Co2+, Mn2+, and Zn2+, and by anions including Cl-, Br-, NO-3, and HCOO-. Neither anions nor divalent cations activated the enzyme without the other. The P-glycolate phosphatase activities from tobacco leaves or the green algae, Chlamydomonas reinhardtii, also required Mg2+ and were activated by chloride. In addition, the enzyme was allosterically inhibited by ribose 5-phosphate. The activation of P-glycolate phosphatase by both anions and divalent cations and the inhibition by ribose 5-phosphate may be involved in the in vivo regulation of P-glycolate phosphatase activity.     

Kinetic and stereochemical studies on reaction mechanism of mouse liver 17 beta-hydroxysteroid dehydrogenases

The kinetic mechanism of two major monomeric 17 beta-hydroxysteroid dehydrogenases from mouse liver cytosol was studied at pH 7 in both directions with NADP(H) and three steroid substrates: testosterone, 5 beta-androstane-3 alpha, 17 17 beta-diol, and estradiol-17 beta. In each case the reaction mechanism of the two enzymes was sequential, and inhibition patterns by-products and dead-end inhibitors were consisted with an ordered bi bi mechanism with the coenzyme binding to the free enzyme, although there was difference in affinity and maximum velocity for the steroidal substrates between the two enzymes. Binding studies of the coenzyme and substrate indicate the binding of coenzyme to the free enzyme, in which 1 mol of NADPH binds to 1 mol of each monomeric enzyme. The 4-pro-R-hydrogen atom of NADPH was transferred to the alpha-face of the steroid molecule by the two enzymes.     

The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent

The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Ribonucleotide reductases: Substrate specificity by allostery

Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.     

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.     

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.     

Pyruvate kinase functions in hot and cold organs of tuna

Summary The skipjack tuna maintains its red skeletal musculature well above ambient temperatures while the temperature of the heart is within 1°C of that of the water. These two tissues exhibit tissue specific forms of pyruvate kinase. The red muscle has one form while the heart has two.TheK _m(PEP) of the red muscle enzymes rises with temperature, within the normal temperature range of the tissue. The affinity of the major form of the heart enzyme for phosphoenolpyruvate is relatively independent of temperature over the physiological temperature range.K _m(ADP) values are temperature independent for both enzymes.Inhibition by alanine of both enzymes is temperature dependent and rises with temperature. The same is true of ATP inhibition of the heart enzyme, but ATP inhibition of the red muscle enzyme is temperature independent. Fructose diphosphate reverses alanine and ATP inhibition at all temperatures.With both enzymes, temperature affects substrate affinities and the sensitivity of the enzyme to metabolite effectors. These effects can be rationalized in terms of physiological significance only in the case of the red muscle enzyme.List of abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - EDTA ethylene diamine tetra acetic acid - FDP fructose 1,6 diphosphate - LDH lactate dehydrogenase - NADH nicotinamide adenine dinucleotide (reduced) - NAD nicotinamide adenine dinucleotide (oxidized) - PEP phosphoenol pyruvate     

Role of substrate inhibition kinetics in enzymatic chemical oscillations.

Two chemical kinetic models are investigated using standard nonlinear dynamics techniques to determine the conditions under which substrate inhibition kinetics can lead to oscillations. The first model is a classical substrate inhibition scheme based on Michaelis-Menten kinetics and involves a single substrate. Only when this reaction takes place in a flow reactor (i.e., both substrate and product are taken to follow reversible flow terms) are oscillations observed; however, the range of parameter values over which such oscillations occur is so narrow it is experimentally unobservable. A second model based on a general mechanism applied to the kinetics of many pH-dependent enzymes is also studied. This second model includes both substrate inhibition kinetics as well as autocatalysis through the activation of the enzyme by hydrogen ion. We find that it is the autocatalysis that is always responsible for oscillatory behavior in this scheme. The substrate inhibition terms affect the steady-state behavior but do not lead to oscillations unless product inhibition or multiple substrates are present; this is a general conclusion we can draw from our studies of both the classical substrate inhibition scheme and the pH-dependent enzyme mechanism. Finally, an analysis of the nullclines for these two models allows us to prove that the nullcline slopes must have a negative value for oscillatory behavior to exist; this proof can explain our results. From our analysis, we conclude with a brief discussion of other enzymes that might be expected to produce oscillatory behavior based on a pH-dependent substrate inhibition mechanism.     

In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. I. Catalytic and regulatory properties of aspartate transcarbamylase

A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharomyces cerevisiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog N-phosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions. Competitive activation as evidence for a catalytic chloride binding site on compound I

Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves

The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.     

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.     

Kinetic analysis of the mechanism of insulin degradation by glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase).

Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.     

New substrates and competitive inhibitors of the Cl- translocating pathway of the uncoupling protein of brown adipose tissue mitochondria

A large number of new substrates for anion uniport by the uncoupling protein of brown adipose tissue mitochondria have been found. These include alkylsulfonates, alkylsulfates and their derivatives, benzenesulfonate, oxohalogenides, hypophosphate, hexafluorophosphate, and pyruvate. Although the spectrum of anion selectivity is far wider than had previously been suspected, there are strong structural requirements for transport. The anion must be monovalent, and polar groups must not be attached to alkyl or aryl chains. The most striking finding is that transport increases dramatically with anion hydrophobicity. Anions that are transported are shown to compete with Cl- for transport by the reconstituted uncoupling protein. For each anion, the Ki for GDP inhibition of transport increases with its rate of transport and correlates inversely with its Ki for competitive inhibition of Cl- transport. For alkylsulfonates, transport rate, Ki for GDP inhibition, and Ki for inhibition of Cl- transport each depend monotonically on alkyl chain length. These findings suggest several new hypotheses relating to the molecular mechanism of transport through uncoupling protein and suggest explanations for observed functional differences among porters belonging to the same gene family.     

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.