scholarly journals A steady-state random-order mechanism for the oxidative deamination of norvaline by glutamate dehydrogenase

1983 ◽  
Vol 211 (1) ◽  
pp. 99-107 ◽  
Author(s):  
C LiMuti ◽  
J E Bell
Keyword(s):  
Steady State ◽  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Order Model ◽  
Oxidative Deamination ◽  
Steady State Kinetics ◽  
Rapid Equilibrium ◽  
Good Agreement

The kinetic mechanism of glutamate dehydrogenase with the monocarboxylic substrate norvaline was examined by using initial-rate steady-state kinetics and inhibition kinetics. To a first approximation the reaction mechanism can be described as a rapid-equilibrium random-order one. Binding synergism between the monocarboxylic substrate and coenzyme is not observed. Dissociation constants for NAD+ and 2-oxoglutarate calculated from the kinetic data assuming a rapid-equilibrium random-order model are in good agreement with independently obtained estimates. Lineweaver-Burk plots with varied norvaline concentration are not strictly linear, and it is concluded that a steady-state random-order model more accurately reflects the observed kinetics with norvaline as substrate.

scholarly journals A product-inhibition study of bovine liver glutamate dehydrogenase

1975 ◽  
Vol 151 (2) ◽  
pp. 305-318 ◽  
Author(s):  
P C Engel ◽  
S S Chen
Keyword(s):  
Glutamate Dehydrogenase ◽  
Competitive Inhibition ◽  
Dissociation Constants ◽  
Random Order ◽  
Bovine Liver ◽  
Product Inhibition ◽  
Rate Equations ◽  
Sodium Phosphate Buffer ◽  
Rapid Equilibrium ◽  
Variable Substrate

1. Initial rates of oxidative deamination of L-glutamate with NAD+ as coenzyme, and of reductive aminiation of 2-oxoglutarate with NADH as coenzyme, catalysed by bovine liver glutamate dehydrogenase were measured in 0.111 M-sodium phosphate buffer, pH 7, at 25 degrees C, in the absence and presence of product inhibitors. All 12 possible combinations of variable substrate and product inhibitor were used. 2. Strict competition was observed between NAD+ and NADH, and between glutamate and 2-oxoglutarate. All other inhibition patterns were clearly non-competitive, except for inhibition by NH4+ with NAD+ as variable substrate. Here the extrapolation did not permit a clear distinction between competitive and non-competitive inhibition. 3. Mutually non-competitive behaviour between glutamate and NH4+ indicates that these substrates can be bound at the active site simultaneously. 4. Primary Lineweaver-Burk plots and derived secondary plots of slopes and intercepts against inhibitor concentration were linear, with one exception: with 2-oxoglutarate as variable substrate, the replot of primary intercepts against inhibitory NAD+ concentration was curved. 5. Separate Ki values were evaluated for the effect of each product inhibitor on the individual terms in the reciprocal initial-rate equations. With this information it is possible to calculate rates for any combination of substrate concentrations within the experimental range with any concentration of a single product inhibitor. 6. The inhibition patterns are consistent with neither a simple compulsory-order mechanism nor a rapid-equilibrium random-order mechanism without modification. They can, however, be reconciled with either type of mechanism by postulating appropirate abortive complexes. Of the two compulsory sequences that have been proposed, one, that in which the order of binding is NADH, NH4+, 2-oxoglutarate, requires an implausible pattern of abortive complex-formation to account for the results. 7. On the basis of a rapid-equilibrium random-order mechanism, dissociation constants can be calculated from the Ki values. Where these can be compared with independent estimates from the kinetics of the uninhibited reaction or from direct measurements of substrate binding, the agreement is reasonable good. On balance, therefore, the results provide further support for the rapid-equilibrium random-order mechanism under these conditions.

scholarly journals Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

111 Validation of the Indicator Amino Acid Oxidation Technique in the Domestic Cat

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 61-61
Author(s):  
Julia Guazzelli Pezzali ◽  
Mahroukh Rafii ◽  
Glenda Courtney-Martin ◽  
Anna-Kate Shoveller
Keyword(s):  
Steady State ◽  
Plasma Concentrations ◽  
Dietary Treatment ◽  
Random Order ◽  
Domestic Cat ◽  
Acid Oxidation ◽  
Linear Regression Models ◽  
Deficient Diet ◽  
Steady State Kinetics ◽  
Adult Cats

Abstract There is a lack of data on the minimum Met or total sulfur AA (SAA) requirements for adult cats. Non-invasive and precise techniques, such as indicator AA oxidation (IAAO) can be used for these purposes. This pilot study aimed to validate the IAAO technique in cats and investigate the effect of dietary Met concentrations and sources on fasted plasma SAA concentrations. Six cats were fed three experimental diets in random order: Met-deficient diet (BASAL; 0.23% Met+Cys-DM) and two Met-sufficient diets in which either DL-Met (DLM) or Met-hydroxy-analogue (MHA) were supplemented, respectively, on an equimolar basis to meet the total SAA requirement (0.34%-DM). After 2d diet adaptation, IAAO studies were performed. Cats were offered 13 ½-hourly small meals. The 6th meal contained a priming dose (4.8 mg/kg-BW) of L-[1-13C]-Phe and the remaining meals the constant dose (1.04 mg/kg-BW). Breath samples were collected ½-hourly to measure 13CO2 enrichment. The following morning after an 16hr fast, blood samples were collected. Isotopic steady-state was evaluated through linear regression models. Plasma AA data were analyzed using PROC GLIMMIX procedure in SAS (Version 9.4). Met concentrations tended to be higher in cats fed DLM compared to BAS (P = 0.0877), but similar to those fed MHA (P > 0.05). Total cysteine plasma concentrations were similar between treatments (P > 0.05). Cats fed BAS had greater concentrations of plasma HCys compared to DLM and MHA (P = 0.0093). Cats did not reach isotopic steady-state in atom percent excess (P >0.05) regardless of the dietary treatment provided. Non-steady-state kinetics models are under development to predict 13CO2 flux. This study indicates that the short-term feeding of a Met-deficient diet in cats results in greater plasma Hcys and a tendency for lower plasma Met concentrations. A higher prime dose of L-[1-13C]-Phe is warranted to achieve isotopic steady-state and successfully apply the IAAO to estimate AA requirements.

scholarly journals Steady-State Kinetic Analysis of Phosphotransacetylase from Methanosarcina thermophila

2006 ◽  
Vol 188 (3) ◽  
pp. 1155-1158 ◽  
Author(s):  
Sarah H. Lawrence ◽  
James G. Ferry
Keyword(s):  
Steady State ◽  
Kinetic Analysis ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Methanosarcina Thermophila ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Acetyl Group ◽  
State Kinetic ◽  
Reversible Transfer

ABSTRACT Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.

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 Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria

1987 ◽  
Vol 244 (2) ◽  
pp. 271-278 ◽  
Author(s):  
R R Ramsay ◽  
J P Derrick ◽  
A S Friend ◽  
P K Tubbs
Keyword(s):  
Glutamate Dehydrogenase ◽  
Gel Filtration ◽  
Random Order ◽  
Bovine Liver ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Purification And Properties ◽  
Rapid Equilibrium ◽  
Variable Substrate ◽  
Myristoyl Coa

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.

scholarly journals Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Yu. Zakharova ◽  
Alexandra A. Kuznetsova ◽  
Victoria I. Uvarova ◽  
Anastasiia D. Fomina ◽  
Liubov I. Kozlovskaya ◽  
...  
Keyword(s):  
Steady State ◽  
Drug Discovery ◽  
Conformational Changes ◽  
Antiviral Drug ◽  
Modern Science ◽  
Kinetic Mechanism ◽  
Wild Type ◽  
Steady State Kinetics ◽  
Main Protease ◽  
Kinetics Of

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre–steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre–steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.

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