Transient-Phase and Steady-State Kinetics for Enzyme Systems Involving Two Substrates

1973 ◽  
Vol 51 (6) ◽  
pp. 832-840 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ternary Complex ◽  
Transient Phase ◽  
Complex Mechanism ◽  
State Equations ◽  
Enzyme Systems ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
The Individual ◽  
Individual Rate

The transient-phase and steady-state equations are derived for four enzyme mechanisms involving two substrates, namely (1) Theorell–Chance mechanism, (2) ping pong bi bi mechanism, (3) ordered ternary-complex mechanism, and (4) random ternary-complex mechanism. In each case, a discussion is presented of the way in which the individual rate constants can be separated on the basis of experimental transient-phase investigations.

scholarly journals Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

2002 ◽  
Vol 184 (15) ◽  
pp. 4096-4103 ◽  
Author(s):  
Yong Ge ◽  
Frédéric H. Vaillancourt ◽  
Nathalie Y. R. Agar ◽  
Lindsay D. Eltis
Keyword(s):  
Steady State ◽  
Pseudomonas Putida ◽  
Sodium Phosphate ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Aromatic Substrate ◽  
Steady State Kinetics ◽  
Full Complement

ABSTRACT Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, k cat = 3.9 ± 0.2 s−1, and K m O2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where K m O2 represents the K m for O2 and KmA represents the K m for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent K m of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.

scholarly journals Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase

2005 ◽  
Vol 386 (2) ◽  
pp. 305-314 ◽  
Author(s):  
Edwin J. A. VELDHUIZEN ◽  
Frédéric H. VAILLANCOURT ◽  
Cheryl J. WHITING ◽  
Marvin M.-Y. HSIAO ◽  
Geneviève GINGRAS ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Km Value ◽  
Substrate Concentrations ◽  
Active Preparation

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3± 0.6 μmol·min−1·mg−1 (20 mM Mes, 80 mM NaCl, pH 6.2, 25 °C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (Km value of 28.6±6.2 μM for HGA, Km value of 1240±160 μM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.

THEORY OF THE TRANSIENT PHASE IN KINETICS, WITH SPECIAL REFERENCE TO ENZYME SYSTEMS

1955 ◽  
Vol 33 (10) ◽  
pp. 1614-1624 ◽  
Author(s):  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Special Reference ◽  
Transient Phase ◽  
Kinetic Behavior ◽  
State Equations ◽  
Enzyme Systems ◽  
Great Excess ◽  
Special Case

The steady-state hypothesis is discussed for enzyme systems, and the conditions under which the steady-state equations will be valid over the main course of the reaction are obtained. It is shown that this is so if the substrate is in great excess, and also under several other circumstances. Equations are derived for the kinetic behavior during the transient phase of the reaction. Two-substrate systems, and the special case of catalase, are considered.

Transient-Phase and Steady-State Kinetics for Inhibited Enzyme Systems. II. Double-Intermediate Mechanisms

1973 ◽  
Vol 51 (6) ◽  
pp. 822-831 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Enzyme Concentration ◽  
Transient Phase ◽  
Enzyme Systems ◽  
Steady State Kinetics ◽  
Inhibited Enzyme

Equations for the pre-steady state and the steady state are derived for enzyme systems in which the enzyme E, the substrate A, and an inhibitor Q are present together, the enzyme concentration being much lower than the concentrations of A and Q. Various mechanisms are considered, ail of them involving two intermediates EA and EA′ (e.g. an acyl enzyme). When the inhibition is reversible the transient phase is followed by the establishment of a steady state. It is shown how experimental pre-steady-state and steady-state results can be analyzed to obtain rate constants, including those for the binding of inhibitor. If the binding of inhibitor is irreversible there is no steady state.

Transient-Phase and Steady-State Kinetics for Inhibited Enzyme Systems. I. Single-Intermediate Mechanisms

1973 ◽  
Vol 51 (6) ◽  
pp. 815-821 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Enzyme Concentration ◽  
Experimental Results ◽  
Transient Phase ◽  
Steady State Analysis ◽  
Noncompetitive Inhibition ◽  
Enzyme Systems ◽  
Steady State Kinetics ◽  
Inhibited Enzyme ◽  
State Analysis

Equations for the pre-steady state and the steady state are derived for enzyme systems in which enzyme E, substrate A, and inhibitor Q are present, the enzyme concentration being lower than the substrate and inhibitor concentrations. It is assumed that the mechanism involves a single intermediate EA. Equations for competitive, anticompetitive, and pure noncompetitive inhibition are derived. When the inhibition is reversible the transient phase is followed by the establishment of a steady state. Analysis of experimental results is discussed for each type of inhibition. If the inhibition is irreversible, there is no steady state.

Transient-Phase and Steady-State Kinetics for Enzyme Activation

1973 ◽  
Vol 51 (6) ◽  
pp. 806-814 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Kinetic Data ◽  
Enzyme Activation ◽  
Transient Phase ◽  
Time Curve ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Substrate Activation ◽  
Steady State Kinetics ◽  
Special Case

A non-steady-state analysis has been worked out for two mechanisms in which an activator Q can become attached to an enzyme–substrate complex EA, the species EAQ breaking down more rapidly than EA. It is shown that if EAQ breaks down into EQ + product there can be no steady state. If, however, EAQ breaks down into E + Q + product, the transient phase is followed by a steady state in which the product versus time curve is linear. A special case of this mechanism is when Q is the substrate (substrate activation). Some published kinetic data on carboxypeptidase are analyzed with reference to the equations derived.

Transient‐phase and steady‐state kinetics of lipase‐catalyzed ester hydrolysis1

1978 ◽  
Vol 13 (2) ◽  
pp. 117-129 ◽  
Author(s):  
M.H. Sadar ◽  
G. Ayranci
Keyword(s):  
Steady State ◽  
Transient Phase ◽  
Steady State Kinetics ◽  
Kinetics Of

Nitrogen and Sulfur Transferases

2007 ◽  
Author(s):  
Perry A. Frey ◽  
Adrian D. Hegeman
Keyword(s):  
Steady State ◽  
Reaction Mechanism ◽  
Active Site ◽  
Reaction Mechanisms ◽  
Radical Reaction ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Polar Reaction

Unlike other group transfer reactions in biochemistry, the actions of nitrogen transferring enzymes do not follow a single unifying chemical principle. Nitrogen-transferring enzymes catalyze aminotransfer, amidotransfer, and amidinotransfer. An aminotransferase catalyzes the transfer of the NH2 group from a primary amine to a ketone or aldehyde. An amidotransferase catalyzes the transfer of the anide-NH2 group from glutamine to another group. These reactions proceed by polar reaction mechanisms. Aminomutases catalyze 1,2-intramolecular aminotransfer, in which an amino group is inserted into an adjacent C—H bond. The action of lysine 2,3-aminomutase, described in chapter 7, is an example of an aminomutase that functions by a radical reaction mechanism. Tyrosine 2,3-aminomutase also catalyzes the 2,3-amino migration, but it does so by a polar reaction mechanism. In this chapter, we consider NH2-transferring enzymes that function by polar reaction mechanisms. Transaminases or aminotransferases are the most extensively studied pyridoxal-5'-phosphate (PLP)–dependent enzymes, and many aminotransferases catalyze essential steps in catabolic and anabolic metabolism. In the classic transaminase reaction, aspartate aminotransferase (AAT) catalyzes the fully reversible reaction of L-aspartate with α-ketoglutarate according to fig. 13-1 to form oxaloacetate and L-glutamate. Like all aminotransferases, AAT is PLP dependent, and PLP functions in its classic role of providing a reactive carbonyl group to function in facilitating the cleavage of the α-H of aspartate and the departure of the α-amino group of aspartate for transfer to α-ketoglutarate (Snell, 1962). PLP in the holoenzyme functions in essence to stabilize the α-carbanions of L-aspartate or L-glutamate, the major biological role of PLP discussed in chapter 3. The functional groups of the enzyme catalyze steps in the mechanism, such as the 1,3-prototropic shift of the α-proton to C4' of pyridoxamine 5'-phosphate (PMP). The steady-state kinetics corresponds to the ping pong bi bi mechanism shown at the bottom of fig. 13-1. This mechanism allows L-aspartate to react with the internal aldimine, E=PLP in fig. 13-1, to produce an equivalent of oxaloacetate, with conversion of PLP to PMP at the active site (E.PMP), the free, covalently modified enzyme in the ping pong mechanism.

scholarly journals Catalysis of potato epoxide hydrolase, StEH1

2005 ◽  
Vol 390 (2) ◽  
pp. 633-640 ◽  
Author(s):  
Lisa T. Elfström ◽  
Mikael Widersten
Keyword(s):  
Steady State ◽  
Epoxide Hydrolase ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Kinetic Mechanism ◽  
Kinetic Measurements ◽  
Steady State Kinetics ◽  
Substrate Conversion ◽  
Km Value ◽  
The Individual

The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 μmol·min−1·mg−1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300→Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

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