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.

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.

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 Modification of pig heart lactate dehydrogenase with methyl methanethiosulphonate to produce an enzyme with altered catalytic activity

1977 ◽  
Vol 161 (3) ◽  
pp. 643-651 ◽  
Author(s):  
D P Bloxham ◽  
D C Wilton
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Thiol Group ◽  
Dilute Solution ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Active Methyl ◽  
Catalytically Active ◽  
Modified Enzyme ◽  
Formation Of Complexes

Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.

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).

scholarly journals Lack of deviation from Michaelis–Menten kinetics for pig heart fumarase

1980 ◽  
Vol 189 (3) ◽  
pp. 653-654 ◽  
Author(s):  
B Andersen
Keyword(s):  
Steady State ◽  
Substrate Concentration ◽  
Substrate Inhibition ◽  
High Substrate Concentration ◽  
High Substrate ◽  
Steady State Kinetics ◽  
Km Value ◽  
Kinetics Of

Studies of steady-state kinetics of fumarase in the usual substrate-concentration range from 0.1 Km to 10 Km and in the high substrate-concentration range from 10 Km to 200 Km are described. The purpose is to investigate reports of substrate inhibition and oscillatory kinetics. In the normal substrate-concentration range, no deviations from hyperbolic kinetics were found, and in the extended concentration range, up to more than 200 times the Km value, no substrate inhibition was demonstrated. A discussion of the discrepancies between the mentioned reports of deviations from the hyperbolic kinetics and the present findings is given.

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