Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

Structure and reaction mechanism of L-rhamnulose kinase from Escherichia coli

Bacterial L-rhamnulose kinase participates in the degradation of L-rhamnose, which is ubiquitous and particularly abundant in some plants. The enzyme catalyzes the transfer of the gamma-phosphate group from ATP to the 1-hydroxyl group of L-rhamnulose. We determined the crystal structures of the substrate-free kinase and of a complex between the enzyme, ADP and L-fructose, which besides rhamnulose is also processed. According to its chainfold, the kinase belongs to the hexokinase-hsp70-actin superfamily. The closest structurally known homologue is glycerol kinase. The reported structures reveal a large conformational change on substrate binding as well as the key residues involved in catalysis. The substrates ADP and beta-L-fructose are in an ideal position to define a direct in-line phosphoryl transfer through a bipyramidal pentavalent intermediate. The enzyme contains one disulfide bridge at a position where two homologous glycerol kinases are regulated by phosphorylation and effector binding, respectively, and it has two more pairs of cysteine residues near the surface that are poised for bridging. However, identical catalytic rates were observed for the enzyme in reducing and oxidizing environments, suggesting that regulation by disulfide formation is unlikely.     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.     

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.     

Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase.

L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose. L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred.     

Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG

MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.     

Studies on the kinetic mechanism and the phosphoryl-enzyme compound of the Escherichia coli acetate kinase reaction.

The Escherichia coli acetate kinase was further examined to determine whether a phosphoryl-enzyme intermediate (or compound) was kinetically discernible or chemically essential in the catalysis of that reaction. The extent of phosphoryl-enzyme formation as monitored by gel filtration of the enzyme-phosphoryl donor substrate complex (E-P) was found to be quite variable. Isolated E-P could be reduced with sodium borohydride and the extent of formation of α-amino-δ-hydroxyvalerate paralleled the observed degree of phosphorylation of the acetate kinase. Incubation with 1 m neutral hydroxylamine of the enzyme, which had been preincubated with acetyl-P for 30–60 min, led to parallel loss of the enzyme's ability to catalyze the net reaction and the ADP ATP exchange reaction. The percentage of activity loss depended upon the concentration of acetyl-P with which the enzyme was preincubated. These observations point toward a requirement for an unmodified active site carboxyl group which may be phosphorylated during the reaction course. The theoretical basis for a new and simple alternative substrate protocol for segregating sequential and ping-pong mechanisms is presented. When applied to acetate kinase, the findings clearly rule out a ping-pong kinetic mechanism. This was also confirmed by initial rate measurements with propionyl-P and ADP, and the weight of the evidence now clearly favors a modified random substrate addition pathway as presented herein. The data do not in any way exclude E-P formation as a requirement for catalysis. Finally, the rates of cold inactivation at 0 °C and reactivation at 29 °C were determined in order to minimize the possible influence of this phenomenon on the kinetic studies.     

Human deoxycytidine kinase: kinetic mechanism and end product regulation

The kinetic properties of the monomeric deoxycytidine kinase (EC 2.7.1.74) from leukemic human T-lymphoblasts have been investigated. The results of steady-state initial-rate kinetic analysis and product inhibition studies at pH 7.5 and 37 degrees C indicate that substrate binding follows an ordered sequential pathway, with the magnesium salt of ATP being the first substrate to bind and dCMP the last product to dissociate. At subsaturating substrate concentrations, dCMP produced competitive inhibition against ATP, while against varied deoxycytidine concentrations dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either substrate. The limiting Km values for deoxycytidine and MgATP were 0.94 and 30 microM, respectively. The end product inhibitor dCTP exhibited competitive inhibition against varied ATP concentration, with a dissociation constant estimated to be 0.7 microM when extrapolated to zero ATP concentration. dCTP was purely noncompetitive against varied deoxycytidine concentration. On the basis of these kinetic results, and on the strong and specific inhibition by dCTP, it is proposed that this end product functions as a multisubstrate analogue, with its triphosphate group binding to the phosphate donor site of the enzyme and its deoxycytidine moiety overlapping and binding to the deoxynucleoside site in a highly specific manner.     

Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.     

A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.     

Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli.

Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [Ki(PEP) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat. Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.     

End-product regulation of carotenogenesis in Phycomyces

Wild-type Phycomyces blakesleeanus synthesizes the yellow pigment, beta-carotene. Colour mutants exhibit various alterations in the biosynthesis of beta-carotene or in its regulation. The presence of certain chemicals in the medium stimulates carotenogenesis in the wild type. We attribute different mechanisms of action to agents which stimulate or fail to stimulate different sets of mutants; this is the case of retinol and dimethyl phthalate. Dimethyl phthalate and veratrol are active on the same mutants, and therefore are likely to act in the same way. The main regulation of carotenogenesis, end-product inhibition, does not operate in the mutants of certain genes; these mutants are indifferent to retinol. By using a collection of retinoids we conclude that their action depends on their structural similarity to a part of the beta-carotene molecule. From these and other observations we propose that end-product inhibition of the pathway is mediated by a complex of beta-carotene and two gene products and that the retinoids compete with beta-carotene and prevent end-product inhibition.Deceased     

Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.     

A kinetic model of oxygen regulation of cytochrome production in Escherichia coli

Recent experimental work has identified the principal components arrayed by Escherichia coli in its sensing of, and response to, varying levels of oxygen. This apparatus may be leveraged/modified by the metabolic engineer to identify nonuniform oxygen and glucose regimens that deliver better yields than their uniform counterparts. Toward this end we build and analyse a mathematical model that captures the role played by oxygen in the regulation of cytochrome production in E. coli.     

Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis

Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex. Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.