Showing papers on "Michaelis–Menten kinetics published in 1971"

Kinetics of the human renin and human substrate reaction.

Abstract: The Michaelis constant (Km) for the hydrolysis of human renin substrate by human renin is 0·931 n-mole/ml. This concentration of substrate is higher than that found in a normotensive population. Since the Km is numerically equal to the concentration of substrate that will permit the reaction to proceed at half its maximal velocity, substrate concentration in normal serum occurs at a very critical level.

Purification and properties of choline acetyltransferase from ox brain striate nuclei.

Abstract: — 1 Choline acetyltransferase was purified from ox brain striate nuclei by an extraction step at pH 5, cation-exchange chromatography, fractional precipitation with ammonium sulphate, and chromatography on Sephadex G-200. The enzyme was obtained free of deacylases and cholinesterases, at specific activities of 01-0-3 μmol acetylcholine formed per min per mg protein. 2 The enzyme was found to be a stable and relatively basic protein, with a molecular weight of 65,000. 3 In the catalysed reactions, , k1, was about four times k2, and the equilibrium constant was approximately 40. For the forward reaction, the Michaelis constant for each substrate was independent of the concentration of the other (choline = 0-75 mM; acetyl-CoA = 10 μM), whereas in the back reaction one substrate increased the affinity for the other (acetylcholine = 0-75-5 MM; CoA = 25-150 μM). 4 CoA inhibited acetylcholine synthesis by competing with acetyl-CoA (K1, = 16 μM). Acetylcholine slightly inhibited the forward reaction (e.g. 45 per cent in 200 mM) without competing with choline or acetyl-CoA. These data indicate an ordered reaction mechanism; acetyl-CoA probably always binds before choline.

Inhibition of Phosphoglycerate Kinase by Products and Product Homologues

Abstract: This paper gives a presentation of ADP and AMP inhibition of phosphoglycerate kinase with MgATP2− and 3-phospho-d-glycerate as substrates at high and low Mg2+ concentrations and pH 7.8. The enzyme seems to contain at least two nucleotide binding sites, one presumably binding to MgATP2− and the other to ADP3−. The ADP3− binding site might bind MgADP1− also. AMP2− competes for the same form of the enzyme, probably the same site, as MgATP2−. ADP3− and MgADP1− are competive inhibitors and AMP2− is a non-competitive inhibitor of 3-P-glycerate. Values of the inhibition constant, Ki, for ADP3− at low Mg2+ level and MgADP1− at high Mg2+ level are 0.2 and 0.02 mM, respectively. The latter value is about ten times less than the expected Michaelis constant for corresponding substrate in the reverse reaction. Ki for AMP is about 2.0 mM at both low and high Mg2+ concentrations but the inhibition is stronger at a high than at a low Mg2+ level, probably caused by conformational and/or other differences of the enzyme at these two metal ion concentrations. The main catalytic reaction suits a pattern that is consistent with a rapid equilibrium random mechanism.

3-Phosphoglycerate Phosphatase in Plants III. Activity Associated with Starch Particles

Abstract: A particulate form of 3-phosphoglycerate phosphatase represents about 20% of this activity in spinach (Spinacia oleracea var. Longstanding Bloomsdale) leaves. By differential and isopycnic sucrose density gradient centrifugation, all the particulate activity was found in starch grains that pelleted through 2.5 m sucrose. This particulate phosphatase was extremely stable, had a pH optimum of 5.8, and an apparent Michaelis constant (3-phosphoglycerate) of 9 x 10(-4)m. No cation requirement for activity could be demonstrated, and the enzyme was inhibited by 0.5 mm Zn(2+) or Cu(2+). The enzyme was most active in catalyzing the hydrolysis of 3-phospho-d-glycerate, but it was not substrate specific. The phosphatase from the starch grains could not be removed by washing, dialysis, homogenization, or treatment with a French pressure cell, but it was solubilized by prolonged sonication or by addition of 0.25 m MgCl(2), when the particles were suspended in 0.8 m sucrose. The solubilized enzyme was partially purified. The properties of the enzyme on the particles or after solubilization were similar to those previously described for the cytosol form of this phosphatase. It is conjectured that the phosphatase of the starch grain regulates glucan synthesis by controlling a 3-phosphoglycerate pool which is an effector for ADP-glucose pyrophosphorylase.

Étude du rameau dégradatif commun des hexuronates chez Escherichia coli K 12

Abstract: The investigation of hexuronate (d-glucuronate and d-galacturonate) metabolism in Escherichia coli K 12 has been completed by studying the biochemical properties of the 2-keto-3-deoxy-6-phosphogluconate aldolase. The enzyme was obtained from a culture of E. coli K 12 grown on galacturonate followed by a 175-fold purification which included these steps: sonic extraction of the enzyme, protamine sulfate precipitation, ammonium sulfate fractionation, heat step and DEAE-Sephadex chromatography. The richest fraction of the chromatographic peak represented 5% of the initial proteins, and has been purified 600-fold. The basic biochemical properties of the enzyme have been established using the DEAE-Sephadex purified extract. The enzyme is stable in a 0.2 M Tris-HCl buffer, pH 7.6; its pH optimum in this buffer is about 7.6. The aldolase seems to function in the absence of coenzyme. In the above-mentioned properties it appears to be related to the enzyme which has been studied in Pseudomonas fluorescens. The Michaelis constant for the substrate 2-keto-3-deoxy-6-phospho-gluconate is about 2 × 10−4 M. Numerous compounds tested have no affinity for this aldolase: only the glyceraldehyde-3-phosphate (substrate of the reverse reaction) and the gluconate-6-phosphate competitively inhibit the aldolase with Ki constants of respectively 4 × 10−4 M and 8 × 10−4 M. The narrow specificity of this aldolase is demonstrated by comparing the compounds with and without affinity for the enzyme; the simultaneous presence of a carboxyl function in position 1 and of a phosphate group in position 6 is necessary for ligand fixation on the enzyme site in the cleavage reaction. The study of denaturation by heat illustrates the thermostability of this enzyme and suggets strongly that at least 99% of the aldolase activity is carried by a homogeneous protein. Half-denaturation is obtained in 10 min at 78°. From the point of view of the physiological regulation, we have shown that the aldolase, which is induced by either of the two hexuronates or by gluconate (the metabolism of which involves the same kind of enzymatic cleavage), is controlled by a single structural gene. Extracts of this aldolase which have been induced separately by each of the three compounds, were purified, mixed and chromatographed on DEAE-Sephadex: the mixture is not dissociable. The kinetics of thermal denaturation of each of these crude extracts are absolutely identical. Finally, the isolation of mutants deficient in aldolase has contributed genetic evidence. These mutants have always simultaneously lost the faculty of metabolizing the three compounds. Spontaneous reversion of these mutants, allowing growth on any of the three substrates, or transduction of any of the three growth phenotypes always leads to the recovery of growth on either of the other two compounds. These different and independent arguments bear out the uniqueness of the aldolase regardless of the inducer employed.

A Nonproteolytic “Trypsin-like” Enzyme Purification and Properties of Arachain

Abstract: By the use of the proteolytic substrates benzoyl-dl-arginine-p-nitroanilide and benzoyl-l-arginine ethyl ester the enzyme arachain has been purified 325-fold from acetone powders of ungerminated peanuts. The pH optimum for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was 8.1 in tris buffer, and for benzoyl-l-arginine ethyl ester was 7.5 using N - 2 - hydroxyethylpiperazine - N' - 2 - ethanesulfonic acid buffer. The purest fraction showed one main band with one to three minor bands on disc gel electrophoresis. The major protein component had an S(20,w) of 6.20. The energy of activation for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was calculated to be 16 kilocalories. The Michaelis constant for benzoyl-dl-arginine-p-nitroanilide was 10 micromolar and for benzoyl-l-arginine ethyl ester was 110 micromolar. The enzyme showed essentially no activity with casein, dimethyl casein, or bovine serum albumin as substrates. A large number of peptides were hydrolyzed by the enzyme, only l-leucyl-l-tyrosine being resistant of the peptides tested. The results suggest that arachain is not a "trypsin-like" protease but is a peptide hydrolase.

Imidazole Catalyses in Aqueous Systems. VIII. Spontaneous Hydrolysis and Michaelis-Menten-type Catalytic Hydrolysis of Phenyl Esters in the Presence of Cholic Acid and Its Histamine Derivative

Abstract: Spontaneous hydrolyses of cationic and anionic phenyl esters in the presence of cholic acid (VI) and their catalytic hydrolyses with a histamine amide of cholic acid (VII) were investigated using a pH-stat at 30°C in 1.0M aqueous KCl. The rates of spontaneous hydrolysis decreased by 30–70% upon binding of substrates with cholic acid. The variation of the apparent binding constant (7-3650M−1) was considered to reflect the magnitude of hydrophobic forces between cholic acid and substrates, although the values may have to be corrected for the possible involvement of self-association of cholic acid molecules. Imidazole derivative VII catalyzed the hydrolysis of phenyl esters according to Michaelis-Menten kinetics. The long methylene chain in the substrate molecule contributed appreciably to the binding of substrate with cholic acid and its derivative. The magnitude of substrate binding with VII appears to be comparable to the true binding constant with cholic acid. The intracomplex rate constants did not diff...