scholarly journals A steady-state approach for inhibition of heterogeneous enzyme reactions

2020 ◽  
Vol 477 (10) ◽  
pp. 1971-1982
Author(s):  
Jeppe Kari ◽  
Corinna Schiano-di-Cola ◽  
Stine Fredslund Hansen ◽  
Silke Flindt Badino ◽  
Trine Holst Sørensen ◽  
...  
Keyword(s):  
Steady State ◽  
Competitive Inhibition ◽  
Industrial Applications ◽  
Rate Equations ◽  
Inhibition Mechanism ◽  
Enzyme Reactions ◽  
Steady State Kinetic ◽  
Solid Liquid Interface ◽  
Solid Liquid

The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid–liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis–Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis–Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

Microstructure Evolution of Ti-Al Peritectic System during the Initiated Stage of Directional Solidification

2007 ◽  
Vol 546-549 ◽  
pp. 1447-1450 ◽  
Author(s):  
Yan Qing Su ◽  
Chang Liu ◽  
Xin Zhong Li ◽  
Jing Jie Guo ◽  
Heng Zhi Fu
Keyword(s):  
Steady State ◽  
Directional Solidification ◽  
Microstructure Evolution ◽  
Aluminum Content ◽  
Solid Phase ◽  
Tial Alloys ◽  
Transient Stage ◽  
Set Up ◽  
Solid Liquid Interface ◽  
Solid Liquid

The microstructure evolution of Ti-Al peretectic system in transient stage and steady state in directional solidification was predicted via theoretical analysis. The solute distribution controlled by diffusion at and ahead the solid-liquid interface will determine whether the properitectic and peritectic phases can nucleate and grow ahead of the opposing solid phase. The formation of banding structure is possible in a certain composition range. At the steady state, a microstructure selection map was set up based on interface response function model. The microstructure of TiAl alloys with different aluminum content was studied with Bridgman directional solidification method. Some evidence in the experiment has been found to support the theoretical prediction.

scholarly journals Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 527-531 ◽  
Author(s):  
A K H MacGibbon ◽  
L F Blackwell ◽  
P D Buckley
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Liver Aldehyde Dehydrogenase

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 × 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.

scholarly journals Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

2010 ◽  
Vol 55 (1) ◽  
pp. 276-283 ◽  
Author(s):  
Jessica A. Brown ◽  
Lindsey R. Pack ◽  
Jason D. Fowler ◽  
Zucai Suo
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

scholarly journals The computerized derivation of rate equations for enzyme reactions on the basis of the pseudo-steady-state assumption and the rapid-equilibrium assumption

1988 ◽  
Vol 251 (1) ◽  
pp. 175-181 ◽  
Author(s):  
H Ishikawa ◽  
T Maeda ◽  
H Hikita ◽  
K Miyatake
Keyword(s):  
Steady State ◽  
Rate Equation ◽  
Rate Equations ◽  
British Library ◽  
Enzyme Reactions ◽  
Input Method ◽  
Steady State Assumption ◽  
Rapid Equilibrium Assumption ◽  
Rapid Equilibrium ◽  
State Assumption

A computer program is developed for the derivation of the rate equation for enzyme reactions on the basis of the pseudo-steady-state assumption and the combination of the pseudo-steady-state and the rapid-equilibrium assumptions. The program not only has an easy input method, but also can obtain a complete rate equation in itself on only one run. The usefulness of the program is demonstrated by deriving the rate equations for some typical enzyme reactions. Details of the program have been deposited as Supplementary Publication SUP 50141 (42 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988), 249, 5.

scholarly journals Ultrasound-based density determination via buffer rod techniques: a review

2013 ◽  
Vol 2 (2) ◽  
pp. 103-125 ◽  
Author(s):  
S. Hoche ◽  
M. A. Hussein ◽  
T. Becker
Keyword(s):  
Industrial Applications ◽  
Temperature Gradients ◽  
Signal Generation ◽  
Temperature Changes ◽  
Density Measurements ◽  
Non Invasive ◽  
Measurement Principle ◽  
High Level ◽  
Solid Liquid Interface ◽  
Solid Liquid

Abstract. The review presents the fundamental ideas, assumptions and methods of non-invasive density measurements via ultrasound at solid–liquid interface. Since the first investigations in the 1970s there has been steady progress with regard to both the technological and methodical aspects. In particular, the technology in electronics has reached such a high level that industrial applications come within reach. In contrast, the accuracies have increased slowly from 1–2% to 0.15% for constant temperatures and to 0.4% for dynamic temperature changes. The actual work reviews all methodical aspects, and highlights the lack of clarity in major parts of the measurement principle: simplifications in the physical basics, signal generation and signal processing. With respect to process application the accuracy of the temperature measurement and the presence of temperature gradients have been identified as a major source of uncertainty. In terms of analytics the main source of uncertainty is the reflection coefficient, and as a consequence of this, the amplitude accuracy in time or frequency domain.

scholarly journals Computer program for the kinetic equations of enzyme reactions. The case in which more than one enzyme species is present at the onset of the reaction

1991 ◽  
Vol 278 (1) ◽  
pp. 91-97 ◽  
Author(s):  
R Varón ◽  
B H Havsteen ◽  
M García ◽  
F García-Canóvas ◽  
J Tudela
Keyword(s):  
Steady State ◽  
Computer Program ◽  
Enzyme System ◽  
Kinetic Equations ◽  
British Library ◽  
Transient Phase ◽  
Enzyme Reactions ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Enzyme Species

This paper presents an extension of the program developed by Varón, Havsteen, García, García-Cánovas & Tudela [(1990) Biochem. J. 270, 825-828] for the expression of the transient-phase and steady-state kinetic equations of a general enzyme system in which the only enzyme species present at the onset of the reaction is the free enzyme. The program has been extended to situations in which more than one enzyme species may be present at the onset of the reaction. The program is given in Supplementary Publication SUP50165 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

scholarly journals Bulgecin A: a novel inhibitor of binuclear metallo-β-lactamases

2005 ◽  
Vol 387 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Alan M. SIMM ◽  
E. Joel LOVERIDGE ◽  
John CROSBY ◽  
Matthew B. AVISON ◽  
Timothy R. WALSH ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Steady State ◽  
Stenotrophomonas Maltophilia ◽  
Competitive Inhibition ◽  
Zinc Ion ◽  
Glycosidic Linkage ◽  
Sugar Moiety ◽  
Aeromonas Veronii ◽  
Steady State Kinetic ◽  
State Kinetic

Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a β-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-β-lactamases. Using steady-state kinetic analysis with nitrocefin as the β-lactam substrate, bulgecin A competitively inhibited the metallo-β-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-β-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-β-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-β-lactamases via the terminal sulphonate group on the sugar moiety.

Effects of modifiers on the kinetics of two-substrate enzyme reactions

1968 ◽  
Vol 46 (11) ◽  
pp. 1381-1396 ◽  
Author(s):  
J. Frank Henderson
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Rate Equations ◽  
Enzyme Reactions ◽  
Ping Pong ◽  
Steady State Rate ◽  
State Rate ◽  
Kinetics Of

Steady state rate equations have been derived for ordered bi bi and ping pong bi bi reactions in which there are (a) one or two nonsubstrate modifiers, (b) two different binding sites for a single nonsubstrate modifier, (c) one or two substrates acting as modifiers, and (d) both nonsubstrate modifiers and substrates acting as modifiers. The deviation of these equations from the Michaelis–Menten equation is shown and methods are suggested by which many of these mechanisms can be distinguished experimentally.

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