scholarly journals Kinetic Analysis Misinterpretations Due to the Occurrence of Enzyme Inhibition by Reaction Product: Comparison between Initial Velocities and Reaction Time Course Methodologies

2021 ◽  
Vol 12 (1) ◽  
pp. 102
Author(s):  
Joana M. C. Fernandes ◽  
Albino A. Dias ◽  
Rui M. F. Bezerra
Keyword(s):  
Kinetic Analysis ◽  
Enzyme Inhibition ◽  
Initial Velocity ◽  
Time Course ◽  
Product Inhibition ◽  
Experimental Point ◽  
Inhibition Effect ◽  
Product Comparison ◽  
Data Points ◽  
Enzymatic Product

The Michaelis–Menten equation (MME) has been extensively used in biochemical reactions, but it is not appropriate when the reaction product inhibits the enzyme. Under these circumstances, each determined initial velocity, v0, is one experimental point that actually belongs to a different MME because enzymatic product inhibition occurs as the reaction starts. Furthermore, the inhibition effect is not constant, since the concentration of the product inhibitor rises as time increases. To unveil the hidden enzyme inhibition and to simultaneously demonstrate the superiority of an integrated Michaelis–Menten equation (IMME), the same range of data points, assuming product inhibition and the presence of a second different inhibitor, was used for kinetic analysis with both methodologies. This study highlights the superiority of the IMME methodology for when the enzyme is inhibited by the reaction product, giving a more coherent inhibition model and more accurate kinetic constants than the classical MME methodology.

Quantitation of measurement error with Optimal Segments: basis for adaptive time course smoothing

1993 ◽  
Vol 264 (6) ◽  
pp. E902-E911 ◽  
Author(s):  
D. C. Bradley ◽  
G. M. Steil ◽  
R. N. Bergman
Keyword(s):  
Measurement Error ◽  
Measurement Errors ◽  
Time Course ◽  
Smooth Curve ◽  
Random Group ◽  
Smooth Curves ◽  
Original Curve ◽  
Wide Range ◽  
Data Points ◽  
Time Courses

We introduce a novel technique for estimating measurement error in time courses and other continuous curves. This error estimate is used to reconstruct the original (error-free) curve. The measurement error of the data is initially assumed, and the data are smoothed with "Optimal Segments" such that the smooth curve misses the data points by an average amount consistent with the assumed measurement error. Thus the differences between the smooth curve and the data points (the residuals) are tentatively assumed to represent the measurement error. This assumption is checked by testing the residuals for randomness. If the residuals are nonrandom, it is concluded that they do not resemble measurement error, and a new measurement error is assumed. This process continues reiteratively until a satisfactory (i.e., random) group of residuals is obtained. In this case the corresponding smooth curve is taken to represent the original curve. Monte Carlo simulations of selected typical situations demonstrated that this new method ("OOPSEG") estimates measurement error accurately and consistently in 30- and 15-point time courses (r = 0.91 and 0.78, respectively). Moreover, smooth curves calculated by OOPSEG were shown to accurately recreate (predict) original, error-free curves for a wide range of measurement errors (2-20%). We suggest that the ability to calculate measurement error and reconstruct the error-free shape of data curves has wide applicability in data analysis and experimental design.

Kinetic mechanism of a recombinant Arabidopsis glyoxylate reductase: studies of initial velocity, dead-end inhibition and product inhibition

2007 ◽  
Vol 85 (9) ◽  
pp. 896-902 ◽  
Author(s):  
Gordon J. Hoover ◽  
Gerald A. Prentice ◽  
A. Rod Merrill ◽  
Barry J. Shelp
Keyword(s):  
Initial Velocity ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Succinic Semialdehyde ◽  
In Planta ◽  
Dead End ◽  
Arabidopsis Protein ◽  
Enzyme Functions ◽  
Glyoxylate Reductase

Kinetic analysis of substrate specificity revealed that a recombinant Arabidopsis protein catalyzes the conversion of glyoxylate to glycolate (Km,glyoxylate = 4.5 μmol·L–1) and succinic semialdehyde (SSA) to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L–1) via an essentially irreversible, NADPH-based mechanism. In this report, the enzyme was further characterized via initial-velocity, dead-end inhibition and product inhibition studies. The kinetic mechanism was ordered Bi Bi, involving the complexation of NADPH to the enzyme before glyoxylate or SSA, and the release of NADP+ before glycolate or γ-hydroxybutyrate, respectively. It can be concluded that the enzyme functions as a NADPH-dependent glyoxylate reductase (EC 1.1.1.79) or possibly an aldehyde reductase (EC 1.1.1.2), and the kinetic mechanism involved is consistent with that found in members of both the aldo-keto reductase and 3-hydroxyisobutyrate dehydrogenase-related superfamilies of enzymes. Since NADP+ was an effective competitive inhibitor with respect to NADPH (Ki = 1–3 µmol·L–1), it is proposed that the ratio of NADPH/NADP+ regulates enzymatic activity in planta.

scholarly journals Development of a Colorimetric Assay and Kinetic Analysis for Mycobacterium tuberculosis D-glucose-1-phosphate Thymidylyltransferase

2011 ◽  
Vol 17 (2) ◽  
pp. 252-257 ◽  
Author(s):  
Shanshan Sha ◽  
Yan Zhou ◽  
Yi Xin ◽  
Yufang Ma
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Kinetic Analysis ◽  
High Throughput Screening ◽  
Initial Velocity ◽  
Colorimetric Assay ◽  
Microtiter Plate ◽  
Kinetic Properties ◽  
Optimal Temperature ◽  
Antituberculosis Drugs ◽  
Optimal Ph

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate–based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.

The Recognition of Glycolate Oxidase Apoprotein with Flavin Analogs in Higher Plants

2004 ◽  
Vol 36 (4) ◽  
pp. 290-296 ◽  
Author(s):  
Wei-Jun Wang ◽  
Jing-Quan Huang ◽  
Chong Yang ◽  
Jiu-Jiu Huang ◽  
Ming-Qi Li
Keyword(s):  
Kinetic Analysis ◽  
Inorganic Phosphate ◽  
Flavin Adenine Dinucleotide ◽  
Brassica Campestris ◽  
Higher Plants ◽  
Competitive Binding ◽  
Glycolate Oxidase ◽  
Flavin Mononucleotide ◽  
Protein Fluorescence ◽  
Inhibition Effect

Abstract The dependence of glycolate oxidase apoprotein (apoGO) activity on flavin analogs was surveyed in 9 higher plants from 7 families. Activities of all apoGOs depended not only on flavin mononucleotide (FMN) but also on flavin adenine dinucleotide (FAD), but not on riboflavin. The kinetic analysis showed that FMN was the optimum cofactor for apoGO from leaves of Brassica campestris. In plant kingdom, FMN, FAD and riboflavin are three flavin analogs with very similar structure, and they could coexist and be inter-converted from each other, so the question is how the apoprotein of glycolate oxidase (GO) recognized these flavin analogs. No inhibition effect of riboflavin on the activity of apoGO with FMN or FAD was found and no obvious quenching of riboflavin or apoGO protein fluorescence was detected with the addition of apoGO or riboflavin, respectively. These results indicated that riboflavin did not bind to apoGO tightly like FMN and FAD. Inorganic phosphate (Pi) did inhibit the activity of GO, and kinetic analysis revealed that this inhibition was caused by the competitive binding to apoGO between Pi and FMN. This competitive binding was further confirmed by the inhibition of Pi to the quenching of FMN and apoGO protein fluorescence with apoGO and FMN, respectively. It was suggested that the 5'-phosphate group of FMN or FAD may play a key role in the recognition and binding of riboflavin analog cofactors with apoGO.

Reactions of ZnR2 Compounds with Dibenzoyl: Characterisation of the Alkyl-Transfer Products and a Striking Product-Inhibition Effect

2011 ◽  
Vol 17 (45) ◽  
pp. 12713-12721 ◽  
Author(s):  
Izabela Dranka ◽  
Marcin Kubisiak ◽  
Iwona Justyniak ◽  
Michał Lesiuk ◽  
Dominik Kubicki ◽  
...  
Keyword(s):  
Product Inhibition ◽  
Inhibition Effect ◽  
Alkyl Transfer

scholarly journals Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade

1993 ◽  
Vol 294 (3) ◽  
pp. 813-819 ◽  
Author(s):  
R Varón-Castellanos ◽  
B H Havsteen ◽  
M García-Moreno ◽  
E Valero-Ruiz ◽  
M Molina-Alarcón ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Kinetic Analysis ◽  
Time Course ◽  
Regulatory Protein ◽  
Steady States ◽  
The State ◽  
Transient Phase ◽  
Effective Sensitivity

A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived.

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.

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