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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
15 Nov 2013-Science
TL;DR: The preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield is reported on, indicating that this elevated pKa results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.
Abstract: Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pK(a) (where K(a) is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mossbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

258 citations

Journal ArticleDOI
TL;DR: The enzyme obeys Michaelis-Menten kinetics in the investigated concentration range, with Km values which are considerably higher than those in bulk water (when concentrations are referred to as water pools), and there is an enhanced turnover number (up to a factor of 6) in micelles with respect to the aqueous solution as discussed by the authors.
Abstract: The enzymatic activity of ..cap alpha..-chymotrypsin solubilized in reverse micelles formed in isooctane by bis(2-ethylhexyl)sodium sulfosuccinate and water (0.6 to 2.5% v:v) has been investigated with the use of n-glutaryl-l-phenylalanine p-nitroanilide as the substrate. The enzyme obeys Michaelis-Menten kinetics in the investigated concentration range, with Km values which are considerably higher than those in bulk water (when concentrations are referred to as water pools). Under certain conditions, there is an enhanced turnover number (up to a factor of 6) in micelles with respect to the aqueous solution. The pH profile of the enzyme activity in the hydrocarbon micellar solution is different from that in water, being shifted to higher pH values and the more so the lower the water content. Under conditions of low water content (0.6 to 1% v:v) the enzyme's stability is greater than in aqueous solution. Structure and activity changes are discussed in terms of the size and structure of the micellar aggregate. 29 references.

258 citations

Journal ArticleDOI
TL;DR: This study presents a method that uses the combination of electrochemical monitoring, chemical analysis, and a titration and off-gas analysis (TOGA) sensor to identify and quantify the sources of electron loss and confirmed the storage of polymeric material in the bacterial cells.
Abstract: Microbial fuel cells (MFCs) are emerging as a novel technology with a great potential to reduce the costs of wastewater treatment. Their most studied application is organic carbon removal. One of the parameters commonly used to quantify the performance of these cells is the Coulombic efficiency, i.e., the electron recovery as electricity from the removed substrate. However, the "inefficiencies" of the process have never been fully identified. This study presents a method that uses the combination of electrochemical monitoring, chemical analysis, and a titration and off-gas analysis (TOGA) sensor to identify and quantify the sources of electron loss. The method was used successfully to close electron, carbon, and proton balances in acetate and glucose fed microbial fuel cells. The method revealed that in the case that a substrate is loaded as pulses carbon is stored inside the cells during initial high substrate conditions and consumed during starvation, with up to 57% of the current being generated after depletion of the external carbon source. Nile blue staining of biomass samples revealed lipophilic inclusions during high substrate conditions, thus confirming the storage of polymeric material in the bacterial cells. The method also allows for indirect measurement of growth yields, which ranged from 0 to 0.54 g biomass-C formed per g substrate-C used, depending on the type of substrate and the external resistance of the circuit.

258 citations

Journal ArticleDOI
TL;DR: Evidence was obtained suggesting that factors other than enzyme or total substrate concentrations affect the velocity of angiotensin formation, and variability of reaction rate may be explained by the existence of an inhibitor or activator in this system or by a variation in the type of substrate.
Abstract: A method is described for estimating plasma renin activity by using renin substrate present in plasma. This method differs from other indirect renin assay methods by (1) incubation in the absence of ions thus establishing conditions for zero order kinetics for the reaction between endogeneous renin and substrate and (2) the use of angiotensinase inhibitors di-sodium ethylenediamine tetraacetic acid (EDTA) and d-isopropylfluorophosphate (DFP). Recoveries of renin added to plasma in levels similar to those occurring in plasma are 85% SD±7%. The incubation was done at pH 5.5 which was shown to be the optimum for human renin reacting with human substrate. By incubating human plasma samples with known quantities of human renin, evidence was obtained suggesting that factors other than enzyme or total substrate concentrations affect the velocity of angiotensin formation. This variability of reaction rate may be explained by the existence of an inhibitor or activator in this system or by a variation in the type of substrate.

257 citations

Journal ArticleDOI
TL;DR: The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity ofThrombin using a microarray of 361 different peptidyl coumarin substrates.
Abstract: A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.

255 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