Evidence for the role of carboxyl groups in activity of endopolygalacturonase of Aspergillus niger. Chemical modification by carbodiimide reagent

1990 ◽  
Vol 55 (5) ◽  
pp. 1389-1395 ◽  
Author(s):  
Lubomíra Rexová-Benková
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Pectic Acid ◽  
Tyrosine Residues ◽  
Group Modification ◽  
Glycine Ethyl Ester ◽  
Decreasing Ph

Endopolygalacturonase (E.C. 3.2.1.15) of Aspergillus niger was modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glycine ethyl ester. The modification resulted in total irreversible inactivation of the enzyme and derivatization of carboxyl acid residues and tyrosine residues. The treatment of the modified enzyme with hydroxylamine led to a restoration of modified tyrosine residues but not to reactivation of the enzyme. The inactivation with carbodiimide was pH dependent, the rate of inactivation increased with decreasing pH. Tri(D-galactosiduronic acid), a competitive inhibitor, or crosslinked pectic acid protected the enzyme against the inactivation. In bioaffinity chromatography of partially inactivated endopolygalacturonase, all residual enzyme activity was retained on the adsorbent while all inactive fraction passed without retardation through the column. On the basis of these results, as well as proximity of the rate constants for enzyme inactivation and the carboxyl group modification it is suggested that the loss of endopolygalacturonase activity is due to the modification of carboxylic acid residues and that at least one is essential for enzyme activity.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

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.

Role of pectic and cellulolytic enzymes in the invasion of the soybean by Rhizobium japonicum

1975 ◽  
Vol 21 (8) ◽  
pp. 1254-1258 ◽  
Author(s):  
William J. Hunter ◽  
Gerald H. Elkan
Keyword(s):  
Enzyme Activity ◽  
Indoleacetic Acid ◽  
Methyl Cellulose ◽  
Rhizobium Japonicum ◽  
Cellulolytic Enzymes ◽  
Pectic Acid ◽  
Pectic Enzyme ◽  
Seedling Inoculation

Past workers have suggested pectic enzyme involvement in the invasion of legumes by Rhizobium. However, no role for pectic acid, pectin, or methyl cellulose depolymerase enzymes in the invasion of R. japonicum was suggested by the current study. Seedling inoculation with infective bacteria did not result in increased enzyme activity. Rhizobium japonicum cell-free culture extracts and 3-indoleacetic acid did not affect the activation, induction, or binding of these enzymes.

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues

1977 ◽  
Vol 55 (8) ◽  
pp. 856-864 ◽  
Author(s):  
T. J. Carne ◽  
T. G. Flynn
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Chemically Modified ◽  
Activity Loss ◽  
Lysyl Residue

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5′-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff s base complex between pyridoxal 5′-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5′-phosphate per mole of PGM dimer through the ε-amino group of a lysyl residue. The effect of pyridoxal 5′-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5′-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5′-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5′-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme.The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

A possible role of arginase in the regulation of polyamine biosynthesis in the rat thyroid

1981 ◽  
Vol 98 (1) ◽  
pp. 57-61 ◽  
Author(s):  
Shigeru Matsuzaki ◽  
Mitsuo Suzuki ◽  
Koei Hamana
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Arginase Activity ◽  
Polyamine Biosynthesis ◽  
Rat Thyroid ◽  
Arginine Concentration

Abstract. Effect of chronic methylthiouracil (MTU) treatment on the thyroid arginase activity and thyroidal concentration of arginine, ornithine and other amino acids was studied in the rat. The activity of thyroid arginase increased significantly at 15 days of MTU treatment and the elevated enzyme activity was reduced to normal by l-thyroxine (T4) injection. The thyroidal concentration of polyamines was increased by MTU and decreased by T4 with the exception of spermine. The thyroidal concentration of arginine and lysine, a substrate and an inhibitor for arginase respectively decreased significantly, while that of ornithine remained unchanged after MTU treatment. T4 injected to MTU-pretreated rats restored the decreased arginine concentration to normal. These results suggest that ornithine supply for polyamine biosynthesis is regulated by the level of both arginase and lysine in the thyroid.

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