Initial Steady-state Velocity Research Articles

High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Analysis of EHL Circular Contact Shut Down

In this paper, an isothermal study of the shut down process of elastohydrodynamic lubrication under a constant load is performed. The surface mean velocity is decreased linearly from the initial steady state value to zero. The details of the pressure and film thickness distributions in the contact area are discussed for the two stages of shut down process, namely the deceleration stage and the subsequent pure squeeze motion stage with zero entraining velocity. The nature of the balance between the pressure, the wedge and the squeeze terms in Reynolds equation enables an analytical prediction of the film thickness change on the symmetry line of the contact in the deceleration period, provided that the steady state central film thickness relationship with velocity is known. The results indicate that for a fixed deceleration rate, if the initial steady state surface mean velocity is large enough, the transient pressure and film thickness distributions in the deceleration period solely depend on the transient velocity. The pressure and film thickness at the end of the deceleration period are then the same and do not depend on the initial steady state velocity. From the same initial steady state velocity, larger deceleration rates provide higher central pressure increase, but also preserve a higher film thickness in the contact area at the end of the deceleration period. Later in the second stage when the axisymmetric pressure and film thickness patterns typical of pure squeeze motion form, the pressure distribution in the contact area resembles a Hertzian contact pressure profile with a higher maximum Hertzian pressure and a smaller Hertzian half contact width. As a result, the film thickness is close to a parabolic distribution in the contact area. The volume of the lubricant trapped in the contact area is then estimated using this parabolic film thickness profile.

Synthesis and evaluation of potential inhibitors of chondroitin AC lyase from Flavobacterium heparinum.

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosaminoglycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Delta4,5-unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1-->4)-alpha-L-threo-hex-4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the "leaving group" sugar moiety. Surprisingly, compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-beta-D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK(a) 8.8. The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid. The anion of compound 2 was found to be a competitive inhibitor with a K(i) value of 5 mM, whereas the conjugate acid had a K(i) value of 35 mM.

Kinetic analysis of cloned cytotoxic T lymphocyte reactivity against normal and leukaemic target cells.

The kinetics of unrestricted killing of normal and leukaemic lymphocyte target cells by a Qa-1b-specific murine cytotoxic T lymphocyte (CTL) clone were evaluated in a manner analogous to enzyme kinetic assays in which the effector and target cells corresponded to the enzyme and substrate, respectively. In order to apply the enzyme-substrate analogy to clonal cytolytic reactions, it was first established that the lytic reactions exhibited initial steady-state velocity of lysis at the effector and target cell concentrations used. The lytic reaction maintained linearity for velocity of lysis during the first 90 min of incubation, then plateaued. Vmax (the maximal rate of target cell lysis achieved by a given effector population) and Km (the target cell number resulting in 1/2Vmax) values were determined over a wide range of target and effector cell concentrations. Both parameters were found to be directly proportional to the number of effector cells. At a given concentration of cloned CTL, the lytic parameters of Vmax and Km were not significantly different for normal or leukaemic target cells that express Qa-1b. Additional kinetic parameters for lysis of normal and leukaemic target cells by a cloned CTL were also compared. The lytic efficiency of the CTL clone (i.e. maximal killing rate with an infinite number of targets) and the intrinsic affinity between effector and target cells were the same with either normal or leukaemic targets. However, the maximal lysis of target cells at an infinite number of effectors was significantly less for normal compared with leukaemic targets. This suggests that the normal target cells were more heterogeneous in their expression of the target (Qa-1b) antigen. Enzyme-like kinetic analysis of cell-mediated lysis reactions can be useful for comparing the relative affinities of effector and target cells obtained from various sources.

Microcomputer simulation of steady-state enzyme kinetics for educational purposes.

A BASIC program to assist the instruction of steady-state enzyme kinetics has been developed for the IBM PC microcomputer. Its purpose is to simulate laboratory experiments in order to minimize the time required to obtain kinetic data from which students deduce kinetic mechanisms and determine kinetic constants of enzyme-catalyzed reactions. The program randomly selects a kinetic scheme from various sequential, ping pong, and iso reaction sequences as well as values for the kinetic constants. The scheme and kinetic constants are unknown to the student at this time; the only thing he or she knows is the stoichiometry of the catalyzed reaction which can have two or three substrates and products. The student is prompted to enter values for concentrations of substrates and products; several different concentrations for each substrate and product can be entered in a single experiment. The program then calculates, displays and prints (if desired) the corresponding initial steady-state velocities. The student can perform as many experiments as desired until enough information is obtained to determine the kinetic mechanism and to calculate values for the kinetic constants.

Elementary steps in the reaction mechanism of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli: kinetics of succinylation and desuccinylation.

The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques. The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate. The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site. The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process. A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1. Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively. At long times, 13-16 mol of succinate binds per mol of unmodified complex. The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes. Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1. The results obtained suggest a limited accessibility between sites on the complex.

A procedure for obtaining initial estimates of parameters appearing in steady-state rate or equilibrium binding equations.

A "peeling" procedure is described for obtaining initial estimates of the parameters in the equation: formula: (see text), where P(x) and Q(x) are polynomials in x. The method is illustrated, in the context of enzyme kinetics, using data which are fitted to the following equation: formula: (see text), where v denotes the initial steady-state velocity at an initial substrate concentration S, and a1, a2, b1, and b2 are non-negative constants. The applicability and limitations of the method for data fitting in fields such as enzyme kinetics and ligand-binding studies are discussed.

Thermodynamics of Enzymatic Transamination of Corticoid-Induced Aminotransferases of F-GOT System in Fowl Blood Plasma : I. Relationship between Reaction Velocity and Substrate Concentration in F-GOT Reaction

Kinetical analysis on the self-control mechanism of corticoid-induced F-GOTgave the following results.Corticoid-induced F-GOT might be one of K class allosteric enzymes and a typical ex-ample of oligomeric enzyme. This enzyme catalyzes the reaction of the forward directionin the presence of L-aspartate and a-ketoglutarate for the following three reasons:(a) F-GOT, particularly homotropic one showing positive cooperativity, shows asigmoid curve in relation to the initial steady-state velocity (Vi) to substrate concentra-tion ([S]), rather than a rectangular hyperbola to be expected from the Michaelis-Mentenrelationship. Apparently, the allosteric transition is also recognized in a cturve of doublereciprocal (Lineweaver-Burk) plot of Vi vs. [S], (b) F-GOT responds to a positive modula-tor with a change in apparent Km(:[84 ;), but without a change in maximum velocity(V=..). This fact proves it to be K enzyme, and (c) Desensitized allosteric F-GOT whichhas lost its previous sensitivity to modulators shows normal Michaelis-Menten kinetics.In this case, a change in V.... in response to the substrate without a change in Michaelisconstant (Km:[5]..), is recognized. This fact proves the catalytic site to be the V form.Such sites are similar to one another. The following consideraiton is put forth: Funda-mentally, F-GOT at a normal steady-state level may exist in a desensitized form ofallosteric F-GOT which has been released by corticoid treatment during the period ofsecondary adaptable responses.Although an excess in substrate resulted in an inhibition of the enzyme activity, thesigmoidal inhibitory curve showed that the kinetic characteristic was not necessarily thesame among enzymes, including an atypical one. It was considered that each enzymemight have been affected differently with its substrate by virtue of its own characteristics.

Prediction of the form of steady-state rate equations from kinetic data

The procedures of fitting kinetic data to initial steady-state rate equations are generally complex when double reciprocal plots of initial velocity vs. initial concentration of reactant (substrate, product or modifier) are not linear. For a general enzyme mechanism, if the initial enzyme concentration and all initial reactant concentrations but one are held constant, then the initial steady-state velocity can be written as a ratio of polynomials in the variable reactant concentration. An examination of some of the properties of the steady-state rate equation for a general enzyme mechanism leads to the proposal of a simple graphical procedure for carrying out preliminary analyses of experimental data. This qualitative procedure, which depends on obtaining initial steady-state velocities at very low and very high reactant concentrations provides a means for determining (1) the difference between the minimum power to which the variable reactant concentration is raised in the numerator polynomial and the minimum power to which it is raised in the denominator polynomial, and (2) the difference between the maximum power to which the variable reactant concentration is raised in the denominator polynomial and the maximum power to which it is raised in the numerator polynomial of the steady-state rate equation. For steady-state rate equations involving quadratic and higher power terms in the variable reactant concentration, this procedure should provide valuable preliminary information on the structure of the rate equation. In addition, the possibility of obtaining quantitative estimates of expressions involving some of the constants appearing in steady-state rate equations is examined.

Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors

A theoretical kinetic study has been undertaken to determine the kinetic characteristics of enzymic reactions that are reversibly inhibited by a substrate analogue at concentrations comparable to that of the enzyme. A general rate equation, that allows for the combination of such a tight-binding inhibitor with one or more enzyme forms of any reaction mechanism, has been derived and shown to be quadratic with square and linear terms in v which represents that true initial steady-state velocity. It has been concluded that, from plots of v against total enzyme concentration at fixed total inhibitor concentration, the concentration of enzyme catalytic sites can be determined irrespective of the nature of the inhibition and the reaction mechanism. However, plots of v( or i /v ) against total inhibitor concentration can be used for this purpose only if the dissociation constant associated with the inhibitor is virtually zero. Particular initial rate equations have been obtained for the cases in which the tight-binding inhibitor gives rise to competitive, non-competitive and uncompetitive inhibition with respect to one of the substrates of a bireactant mechanism. From these equations it has been deduced that, in the presence of inhibitor, plots of i /v against the reciprocals of the substrate concentrations yield families of concave-down, non-rectangular hyperbolas. The difficulties associated with the determination of inhibition constants for tight-binding inhibitors are discussed.

Transient phase kinetics of enzyme reactions

A solution is presented for the transient phase of a general enzyme mechanism and some of its properties are examined. The solution for the concentrations of the reactant components (substrates, products and modifiers) of the mechanism as functions of time asymptotically approach a straight line as time approaches infinity. Expressions are obtained for the slope and intercept on the time axis of this straight line. Both these parameters (slope and intercept on the time axis) can be written as a ratio of polynomials in the initial reactant concentrations, the coefficients being functions of the velocity constants of the proposed mechanism (the slope is the usual initial steady-state velocity expression for the reaction). These two parameters provide a means of differentiating among various proposed mechanisms.

Initial Steady State Velocities in the Evaluation of Enzyme-Coenzyme-Substrate Reaction Mechanisms.

Initial Steady State Velocities in the Evaluation of Enzyme-Coenzyme-Substrate Reaction Mechanisms.