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Substrate (chemistry)

About: Substrate (chemistry) is a research topic. Over the lifetime, 35902 publications have been published within this topic receiving 740722 citations. The topic is also known as: enzyme substrate.


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Journal ArticleDOI
TL;DR: These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.
Abstract: The bacterial phosphotriesterase has been utilized as a template for the evolution of improved enzymes for the catalytic decomposition of organophosphate nerve agents. A combinatorial library of active site mutants was constructed by randomizing residues His-254, His-257, and Leu-303. The collection of mutant proteins was screened for the ability to hydrolyze a chromogenic analogue of the most toxic stereoisomer of the chemical warfare agent, soman. The mutant H254G/H257W/L303T catalyzed the hydrolysis of the target substrate nearly 3 orders of magnitude faster than the wild-type enzyme. The X-ray crystal structure was solved in the presence and absence of diisopropyl methyl phosphonate. The mutant enzyme was ligated to an additional divalent cation at the active site that was displaced upon the binding of the substrate analogue inhibitor. These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.

125 citations

Journal ArticleDOI
TL;DR: Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics and initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism.

125 citations

Journal ArticleDOI
TL;DR: A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.
Abstract: The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.

125 citations

Journal ArticleDOI
TL;DR: The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated, and results show that the specific role of calcium in all the events of the catalysttic cycle at the active site is not shared by other divalentCations.
Abstract: The divalent cation requirement for partial reactions of the catalytic turnover cycle during interfacial catalysis by pig pancreatic phospholipase A2 (PLA2) is investigated. Results show that the specific role of calcium in all the events of the catalytic cycle at the active site is not shared by other divalent cations. Cations such as calcium, barium, and cadmium bind to the enzyme in the aqueous phase. The active-site-directed ligands (substrate, products, and transition-state mimics) do not bind to the enzyme in the absence of a divalent cation. The synergistic binding of such ligands to the active site of PLA2 bound to the interface is, however, observed only in the presence of isosteric ions like calcium and cadmium, but not with larger ions like strontium or barium. The equilibrium constants for ligands bound to the enzyme in the presence of calcium and cadmium are virtually the same. However, only calcium supports the catalytic turnover; the rate of hydrolysis in the presence of cadmium is less than 1% of that observed with calcium. The role of divalent ions on the interfacial catalytic turnover cycle of PLA2 is not only due to the cation-assisted binding of the substrate but also due to its participation in the chemical step. Other roles of divalent ions in the events of interfacial catalytic turnover are also identified. For example, the binding of the enzyme to the interface is apparently promoted because the divalent cation is required for the sequential step, i.e., the binding of the substrate to the active site of PLA2.(ABSTRACT TRUNCATED AT 250 WORDS)

125 citations

Journal ArticleDOI
09 Sep 2013-ACS Nano
TL;DR: A weak attractive interaction between substrate molecules and the scaffold creates a "virtual compartment" and substantially accelerates initial throughput and if intermediate substrates can diffuse freely, placing individual enzyme pairs on scaffolds is only beneficial in large cells, unconfined extracellular spaces or in systems with sequestering reactions.
Abstract: The concept of "metabolic channeling" as a result of rapid transfer of freely diffusing intermediate substrates between two enzymes on nanoscale scaffolds is examined using simulations and mathematical models. The increase in direct substrate transfer due to the proximity of the two enzymes provides an initial but temporary boost to the throughput of the cascade and loses importance as product molecules of enzyme 1 (substrate molecules of enzyme 2) accumulate in the surrounding container. The characteristic time scale at which this boost is significant is given by the ratio of container volume to the product of substrate diffusion constant and interenzyme distance and is on the order of milliseconds to seconds in some experimental systems. However, the attachment of a large number of enzyme pairs to a scaffold provides an increased number of local "targets", extending the characteristic time. If substrate molecules for enzyme 2 are sequestered by an alternative reaction in the container, a scaffold can result in a permanent boost to cascade throughput with a magnitude given by the ratio of the above-defined time scale to the lifetime of the substrate molecule in the container. Finally, a weak attractive interaction between substrate molecules and the scaffold creates a "virtual compartment" and substantially accelerates initial throughput. If intermediate substrates can diffuse freely, placing individual enzyme pairs on scaffolds is only beneficial in large cells, unconfined extracellular spaces or in systems with sequestering reactions.

125 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202214
2021807
20201,053
20191,064
20181,112
20171,024