scholarly journals Modification of an arginine residue in pig kidney general acyl-coenzyme A dehydrogenase by cyclohexane-1,2-dione

1982 ◽  
Vol 207 (3) ◽  
pp. 415-419 ◽  
Author(s):  
Z Y Jiang ◽  
C Thorpe
Keyword(s):  
Residual Activity ◽  
Competitive Inhibitor ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Excess Substrate ◽  
Arginine Residues ◽  
Modified Enzyme ◽  
High Concentrations ◽  
Acyl Coenzyme A

The flavoenzyme pig kidney general acyl-CoA dehydrogenase (EC 1.3.99.3) is inactivated by cyclohexane-1,2-dione in borate buffer in a reaction that exhibits pseudo-first-order kinetics. Strong protection is afforded by the substrate octanoyl-CoA, as well as by heptadecyl-CoA, a potent competitive inhibitor of the dehydrogenase that does not reduce enzyme flavin. Enzyme exhibiting 10% residual activity in borate buffer contains about 1.3 modified arginine residues per flavin molecule. Very little reduction of the modified enzyme in borate buffer occurs at high concentrations of octanoyl-CoA, in marked contrast with the stoicheiometric reduction of the native enzyme. However, in phosphate buffer alone, the modified enzyme exhibits 55% residual activity and, although binding of substrate is still seriously impaired (apparent Kd=14 microM), excess substrate effects the formation of the characteristic reduced flavin X enoyl-CoA charge-transfer complex. These results suggest that the susceptible arginine residue, though not catalytically essential, is probably within the acyl-CoA-binding site of general acyl-CoA dehydrogenase.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

scholarly journals The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 137 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Owen A. Young ◽  
John W. Anderson
Keyword(s):  
Fatty Acid ◽  
Pinus Radiata ◽  
Kinetic Studies ◽  
Single Species ◽  
Competitive Inhibitor ◽  
Fatty Acyl ◽  
Short Chain ◽  
Low Concentrations ◽  
High Concentrations ◽  
Acyl Coenzyme A

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PPi–ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP2O72- but not with the concentration of Mg2+, as judged by PPi–ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PPi. In PPi–ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PPi–ATP exchange. PPi–dATP exchange was less than PPi–ATP exchange at low concentrations of fatty acid, but at higher concentrations PPi–dATP exchange exceeded PPi–ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PPi. The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PPi. The inhibition by propionate was non-competitive with respect to propionyl-AMP and PPi. 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PPi–ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PPi is probably the only product released.

scholarly journals Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5′-phosphate

1978 ◽  
Vol 173 (1) ◽  
pp. 53-58 ◽  
Author(s):  
I A Hemmilä ◽  
P I Mäntsälä
Keyword(s):  
Enzyme Activity ◽  
Glutamate Dehydrogenase ◽  
Bacillus Megaterium ◽  
Glutamate Synthase ◽  
Complete Loss ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Complete Protection ◽  
Complete Inactivation ◽  
Arginine Residues

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5′-phosphate and this inactivation increased 3–4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5′-phosphate.

scholarly journals Evidence for the presence of an essential arginine residue in photoreactivating enzyme from Streptomyces griseus

1985 ◽  
Vol 229 (2) ◽  
pp. 469-476 ◽  
Author(s):  
A P M Eker
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Streptomyces Griseus ◽  
Enzymic Activity ◽  
Borate Buffer ◽  
Arginine Residue ◽  
Dna Binding Site ◽  
Arginine Residues ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Butane-2,3-dione inhibits the enzymic activity of Streptomyces griseus photoreactivating enzyme (PRE). Some characteristics of the inhibition, notably the enhancement by borate buffer and the reversibility, indicate that arginine residues are modified. From the kinetics of inhibition it can be concluded that a single essential arginine residue is involved. U.v.-irradiated DNA, the substrate for PRE, protects the enzyme against inactivation by butane-2,3-dione. This suggests that the essential arginine residue is situated in or near the u.v.-irradiated-DNA-binding site. Non-irradiated DNA at higher concentrations also protects against inactivation, indicating that PRE can form non-specific complexes. From the ratio of complex constants obtained from protection experiments with non-irradiated and u.v.-irradiated DNA it appears that PRE preferably binds to dimer sites.

scholarly journals The Sustainable Production of a Novel Laccase from Wheat Bran by Bordetella sp. JWO16: Toward a Total Environment

Catalysts ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 677
Author(s):  
John Onolame Unuofin
Keyword(s):  
Wheat Bran ◽  
Contaminated Soils ◽  
Residual Activity ◽  
Dye Decolorization ◽  
Fermentation Medium ◽  
Biochemical Properties ◽  
Synthetic Dyes ◽  
High Concentrations ◽  
Capital Intensive ◽  
Agroindustrial Residue

Laccase is increasingly adopted in diverse industrial and environmental applications, due to its readily accessible requirements for efficient catalytic synthesis and biotransformation of chemicals. However, it is perceived that its industrial production might incur some unfavorable overhead, which leads to expensive market products, and the corresponding negative environmental feedback, due to the use of capital-intensive and precarious chemicals. To this end, this study was designed to evaluate the performance indicators of the valorization of wheat bran by a novel Jb1b laccase and its subsequent application in waste minimization and water management, on a laboratory scale. Optimal Jb1b laccase was produced in submerged fermentation medium containing wheat bran, an agroindustrial residue, through response surface methodology (RSM) algorithm, and was applied in dye decolorization and denim bioscouring, respectively. Results showed that the resultant enzyme manifested unique biochemical properties, such as enhanced tolerance at certain physicochemical conditions, with a residual activity of at least ca. 76%. Furthermore, phenomenally high concentrations of synthetic dyes (0.2% w v−1) were decolorized over 56 h, and a 6 h mediator-supported simultaneous denim bleaching and decolorization of wash effluent was observed. The sustainability of the production and application processes were inferred from the reusability of the fermentation sludge as a potential biofertilizer, with subsequent prospects for the biostimulation and bioaugmentation of contaminated soils, whereas the decolorized water could be adopted for other uses, amongst which horticulture and forestry are typical examples. These phenomena therefore authenticate the favorable environmental feedbacks and overhead realized in this present study.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

Interaction of acyl coenzyme A substrates and analogs with pig kidney medium-chain acyl-CoA dehydrogenase

Biochemistry ◽  
1987 ◽  
Vol 26 (12) ◽  
pp. 3704-3710 ◽  
Author(s):  
Patricia J. Powell ◽  
Sze Mei Lau ◽  
David Killian ◽  
Colin Thorpe
Keyword(s):  
Coenzyme A ◽  
Medium Chain ◽  
Pig Kidney ◽  
Chain Acyl ◽  
Acyl Coenzyme A

scholarly journals Role of Arginines in Coenzyme A Binding and Catalysis by the Phosphotransacetylase from Methanosarcina thermophila

2001 ◽  
Vol 183 (14) ◽  
pp. 4244-4250 ◽  
Author(s):  
Prabha P. Iyer ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Sulfhydryl Group ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Methanosarcina Thermophila ◽  
Arginine Residues ◽  
Substrate Protection

ABSTRACT Phosphotransacetylase (EC 2.3.1.8 ) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH3COOPO3 2− + CoASH ⇆ CH3COSCoA + HPO4 2−. The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in k cat andk cat/Km for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases ink cat andk cat/Km were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.

scholarly journals The human complement system: assembly of the classical pathway C3 convertase

1980 ◽  
Vol 189 (1) ◽  
pp. 173-181 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Direct Evidence ◽  
Gel Filtration ◽  
Fluid Phase ◽  
Classical Pathway ◽  
Human Complement ◽  
Stabilizing Effect ◽  
Excess Substrate ◽  
High Concentrations ◽  
Low Ionic Strength

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

scholarly journals Glycation of the human erythrocyte glucose transporter in vitro and its functional consequences

1990 ◽  
Vol 268 (3) ◽  
pp. 661-667 ◽  
Author(s):  
P J Bilan ◽  
A Klip
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Competitive Inhibitor ◽  
Human Erythrocyte Membrane ◽  
Functional Consequences ◽  
High Concentrations ◽  
Erythrocyte Membrane Proteins

Glycation of human erythrocyte membrane proteins was induced by incubation in vitro with high concentrations (80 mM or 200 mM) of D-glucose for 3 or 6 days. The extent of glycation was quantified from the covalent incorporation of 3H by reduction of the glucose glycation products with NaB3H4. For membranes incubated for 3 days with 80 mM-D-glucose, glycation in vitro of Band 4.5 (containing the glucose transporter) was equivalent to 0.11 mol of glucose/mol of glucose transporter, compared with 3H labelling in 3-day-incubated control membranes of 0.055 mol of glucose/mol of glucose transporter. In membranes incubated for 6 days with 200 mM-D-glucose, glycation increased to 0.21 mol of glucose/mol of glucose transporter, whereas the controls without glucose had 0.11 mol of glucose/mol of glucose transporter. Glycation in vitro was accompanied by a fall in the Bmax of binding of [3H]cytochalasin B (a competitive inhibitor of glucose transport), without any change in the binding affinity. The data suggest that glycated glucose transporters have decreased ability to bind cytochalasin B. It is proposed that glycation can alter glucose transporter activity.

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