Showing papers on "Michaelis–Menten kinetics published in 1985"

Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies.

Abstract: The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pKa of 7.3. The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1. Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor. The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.

Oxidation of ethylene by cotyledon extracts from Vicia faba L. : Cofactor requirements and kinetics.

Abstract: Improved rates of ethylene oxidation by cell-free preparations from cotyledons of Vicia faba L. have been obtained using cryogenic storage techniques and by developing a method for the hydrolysis of ethylene oxide. Gel permeation chromatography showed that a low-molecular-size fraction was required for activity; accordingly, the kinetics of ethylene oxidation in the presence of this fraction were studied. Reduced pyridine nucleotides could substitute for the low-molecular-size fraction. Activity under a nitrogen atmosphere was 60% lower than in air. The need for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen indicated that the enzyme might be a mixed-function oxidase. Using sufficient NADPH to approach saturation, the apparent Michaelis constant (Km) for ethylene was 1.94±0.38 · 10-8 M (aqueous phase), and when ethylene was saturating, the Km for NADPH was 3.7 · 10-5 M. Carbon monoxide was found to inhibit by competing with ethylene, and the inhibitor constant was 5.97 · 10-7 M in solution. In the presence of excess ethylene and NADPH, activity was highest in phosphate-buffered medium pH 7.9. The bulk of the activity was found in a microsomal fraction.

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris

Abstract: ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the Vmax. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half Vmax value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half Vmax value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn2+ and Mg2+ were highly sigmoidal. The activator concentration at half Vmax values for Mn2+ and Mg2+ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.

Influence of pH on the regulatory kinetics of rat liver phosphofructokinase: a thermodynamic linked-function analysis.

Abstract: The relationship between pH and the MgATP inhibition of rat liver phosphofructokinase has been quantiatively evaluated by utilization of a thermodynamic linked-function approach This approach obviates the need to presuppose discrete inhibited and active states of the enzyme The behavior of the apparent Michaelis constant for fructose 6-phosphate (Fru-6-P) over a 100-fold concentration range of MgATP conforms to the behavior predicted by the linked-function theory in that, a high concentrations of MgATP, saturation of the inhibitory effect is achieved, a result not predicted by a mutually exclusive two-state model This behavior is described by the relationship Ka = Ka0[(Kix0 + [X])]/(Kix0 + Q[X])], where Ka is the apparent Michaelis constant for Fru-6-P, Ka0 is the Michaelis constant for Fru-6-P in the absence of MgATP, Kix0 is the dissociation constant of MgATP in the absence of Fru-6-P, and Q is the coupling term that quantitatively describes the finite degree of antagonism between MgATP and Fru-6-P The free energy of interaction between MgATP and Fru-6-P, obtained from Q, is 19 kcal/mol at 25 degrees C Ka0 and Kix0 are 017 and 03 mM, respectively The influence of pH on these three parameters was then systematically investigated, and only Ka0 increased substantially with decreasing pH Consequently, it is concluded that decreasing the pH does not increase the apparent Ka for Fru-6-P by augmenting the binding or inhibition by MgATP to a significant extent but rather by directly affecting the intrinsic affinity of the enzyme for Fru-6-P The pK for this effect is 81

ADP Is a Competitive Inhibitor of ATP-Dependent H+ Transport in Microsomal Membranes from Zea mays L. Coleoptiles

Abstract: An anion-sensitive ATP-dependent H(+) transport in microsomal membranes from Zea mays L. coleoptiles was partially characterized using the pH gradient-dependent decrease of unprotonated neutral red. The following criteria strongly suggest a tonoplast origin of the H(+) transport observed: strict dependence on Cl(-); inhibition by SO(4) (2-) and NO(3) (-); insensitivity against vanadate, molybdate, and azide; reversible inhibition by CaCl(2) (H(+)/Ca(2+) antiport); inhibition by diethylstilbestrol. The substrate kinetics revealed simple Michaelis Menten kinetics for ATP in the presence of 1 millimolar MgCl(2) with a K(m) value of 0.56 millimolar (0.38 millimolar for MgATP). AMP and c-AMP did not influence H(+) transport significantly. However, ADP was a potent competitive inhibitor with a K(i) value of 0.18 millimolar. The same inhibition type was found for membranes prepared from primary roots by the same procedure.

New and simple method to predict dosage of drugs obeying simple Michaelis-Menten elimination kinetics and to distinguish such kinetics from simple first order and from parallel Michaelis-Menten and first order kinetics

Abstract: It has been found empirically that either average or minimum steady-state plasma concentrations (Css) of drugs obeying Michaelis-Menten elimination kinetics give essentially linear plots on semilogarithmic graph paper when Css is plotted versus the maintenance dose (D) or dose rate (R). The equations of such straight lines may be converted to the following nonlinear equation: Css = abD which fits the Css,D data essentially as well as D = VmCss/(Km + Css). The parameter b is analogous to unity plus the interest fraction in logarithmic growth or compound interest calculations, and each drug appears to have a characteristic value of this parameter, with extremely small intersubject variation. From the above equation the following equation, Dn+1 = Dn + 1n(Cn+1/Cn)1n b can be derived, which forms the basis of predicting the needed dosage, Dn+1, to obtain a desired steady-state concentration, Cn+1, using one initial steady-state concentration, Cn, obtained with dose, Dn, and using a population value of b for the drug. It appears that it is the value of the "initial capital" (i.e., a in relation to the initial dose) rather than the "interest fraction" (i.e., b - 1) that causes most of the intersubject variation in Css of a given drug. Several drugs illustrate the usefulness of the method. A semilogarithmic plot also appears to be an excellent method to distinguish simple Michaelis-Menten kinetics from parallel Michaelis-Menten and first order elimination kinetics and from simple first order kinetics with steady-state data in the range 0.3-3 Km.

Characterization and comparison of soluble and immobilized pig muscle aldolases.

Abstract: Pig muscle aldolase was insolubilized by covalent attachment to a polyacrylamide matrix containing carboxylic functional groups. The catalytic activity of the Akrilex C-aldolase was 2014 units/g solid, i.e., an activity loss of only about 5% relative to the initial activity. The pH optimum for catalytic activity shifted form 7.25 to 7.5 and the apparent temperature optimum from 313 to 318 K. The Michaelis constant of the insolubilized enzyme was significantly higher than that of the soluble aldolase. Heat- and urea-inactivation experiments revealed that the immobilization increased the stability of the enzyme.

Active Center and Mode of Reaction of Blasticidin S Deaminase

Abstract: Blasticidin S deaminase (EC 35423) was purified by affinity chromatography using a column of Sepharose 4B coupled with pyrimidinoblasticidin S as a ligand The Michaelis constant Km and the maximum velocity Vmax varied with pH, which suggests that enzyme ionization is important either for substrate binding or for catalytic activity The inflection point at pH 86-89 in the plot of pKm versus pH was attributed to an enzyme amino group which corresponded to the carboxyl group of substrates, and an inflection at pH 53 in the log Vmax-pH plot was assigned to the imidazole group of histidine, which appeared to be critical for catalytic deaminohydroxylation The binding of substrates by the enzyme was inferred to be promoted thermodynamically, and activation of the substrate-enzyme complex was presumed to proceed endothermically From the results obtained, a hypothetical mode of reaction for blasticidin S deaminase is proposed

Effects of pH and of Temperature on Saturable Transport Processes

Abstract: In their molecular nature, specific membrane transport processes resemble enzyme reactions taking place in a continuous phase: They involve binding of a “substrate” molecule to a specific receptor site, the translocation proper corresponding to the substrate → product conversion, and dissociation of the “product”, in the transport case simply release of the bound molecule or ion to another aqueous phase, separated from the starting aqueous phase by the membrane. The amount of evidence supporting the above sequence of events is vast and need not be reiterated here. Kinetically, all such transports are characterized by a half-saturation constant which is formally identical with the Michaelis constant of enzyme kinetics and is a similarly complicated function of various rate constants comprised in the mechanism, and by a maximum rate of transport, involving the total amount of carrier protein present in a given amount of cells and a combination of first-order rate constants, again depending on the complexity of the system under consideration.

Study of immobilized yeast, saccharomyces cerevisiae, using flow microcalorimetry

Abstract: When immobilized yeast was exposed to nutrients, the resulting heat effect (dQ/dt; J sec-1) increased exponentially with a doubling time (t2) of 2.2±0.3hr. The half life (dQ/dt) under non-growing conditions with sucrose as substrate was 84hr. The kinetics of the transformation of a series of sugars were characterised. The Michaelis constant (Km) and maximal heat effect, (dQ/dt)max, were determined using two common enzyme kinetics linearization plots. The shapes of the Eadie plots for some sugars are discussed in terms of currently proposed mechanisms of their uptake

Micro-computer Analysis of Enzyme-catalyzed Reactions by the Michaelis-Menten Equation

Abstract: Although Bardsley et al.1) theoretically and experimentally found that, in a very wide range of substrate concentrations, all enzymes probably deviate from the Michaelis-Menten equation and need equations of higher degrees, most enzyme kinetics have still been regarded to be Michaelian without serious limitations, and their kinetic constants such as the Michaelis constant (Km), maximum velocity (Vmax), and inhibition constant (Ki) have been calculated largely from the initial velocity data by the Lineweaver-Burk plot. Accordingly the poor reliability and reproducibility of Km and Vmax have widely been recognized for many enzymes, but an effective remedy has not yet been devised. To improve the present state of enzyme kinetics, we propose a practical method in which, with a micro-computer, an assay data set of an enzyme is critically checked at each data point for reasonable application of the differential Michaelis-Menten equation and then subjected to the calculation of Km and Vmax estimates by 5 methods. In particular, the assay data set composed of substrate concentration ([S0]) and initial reaction velocity (v) are corrected for substrate consumption, preferably by the method of Lee and Wilson.2) The whole data set is then checked for applicability of the Michaelis-Menten equation by three linear plots [Lineweaver-Burk (L-B), Hanes-Woolf (H-W), and Scatchard (SCAT) (or Eadie-Hofstee) plots] so that an unacceptable data pair(s), if present, can reasonably be deleted by visual examination. The trimmed data set is finally used for calculation of Km and Vmax by the three linear plots, the Wilkinson (WILK) method, and the nonparametric (NONP) method (or direct linear plot).

Structure and properties of enzyme graft copolymers: Temperature effects on HRP immobilization mechanism.

Abstract: The possibility of obtaining immobilized horseradish peroxidase (HRP) materials with K'(m) values close to that of the native enzyme, but with good thermal stability, was investigated. The photochemical reaction was used as the immobilization methodology. Temperature and catalyst concentration were found to be the main parameters able to control the immobilization reaction mechanism more than type of functional monomer, polymer-matrix, and enzyme-polymer ratios. By carrying out the immobilization reaction at 35 degrees C and using either bisacryloylpiperazine (BAP) or hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as the functional monomer, materials with a good thermal stabilization (the retained activity after 240 min at 60 degrees C was between 65-25%) as well as kinetic constants (0.6-0.8 x 10(-4)M) similar to that of the free enzyme (0.57 x 10(-4)M) were obtained. Since low K'(m) values were obtained also using a high polymer content (pBAP copolymers, 25%; pHTsT copolymers, 30%) and neither limitation to substrate diffusion nor a reduction of the enzyme mobility was found, the enzyme should be linked to the matrix during the last steps of monomer polymerization, and it should have an external disposition with respect to the support.

Effectiveness factor suffering from decrease of surface area caused by compaction of immobilized enzyme particles in packed columns. Case of the michaelis-menten type reaction.

Abstract: 充填層型反応器において圧密が生じている場合の固定化酵素粒子内の拡散を数値的に調べた.各粒子が複数の密着部を持つとして, 1個の密着部を含む部分領域に注目した2次元拡散モデルを提案し, Michaelis-Menten型反応に従う場合の濃度分布を有限差分法と有限要素法を用いて解析した.有効係数における圧密効果を評価した結果, 有効係数は, 密着面積とgeneral modulusの増加に伴って減少することがわかった.また有効係数に対する近似式が従来報告された小林らの方法の拡張により取扱い可能であることを示した.

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella

Abstract: ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris lbh. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-I-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V,,.,^. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V,,,, value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-l-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V.. value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn2 and Mg2" were highly sigmoidal. The activator concentration at half V,,,,, values for Mn2" and Mg2 were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.