Km For Reaction Research Articles

Thermokinetic Studies on the Activation of Arginase by Glycine

AbstractThe activation of bovine liver arginase, which catalyzes the hydrolysis of L‐arginine to L‐ornithine and urea, by glycine was studied by thermokinetic methods at 37°C in 40 mmol·L−1 sodium barbiturate‐HCl buffer solution (pH 9.4). Results of this experiment indicate that an appropriate concentration of glycine can enhance the activity of arginase, and the relative activation rate reached its maximum value, 74%, when the concentration of glycine in reaction system was 1 mmol·L−1 and the initial concentration of arginine was 5 mmol·L−1. With the increase of substrate concentration, the relative activation rate decreased in a definite glycine concentration. Michealis constant Km of reaction decreased from 5.53 to 3.31 mmol·L−1 and inhibition constant of product L‐ornithine Kp increased from 1.18 to 3.73 mmol·L−1 when glycine concentration was 1 mmol·L−1. For these reasons one possible activation mechanism of arginase by glycine was suggested that the activation effect results from the competition of glycine and arginine to enzyme activity position. When one or two of the activity positions of arginase are occupied by glycine, it is propitious for the enzyme to complex with substrate and obstruct L‐ornithine from combining with enzyme, and when all of the activity positions are occupied by glycine, the activation effect vanishs and the inhibition effect appears.

Mae inhibits Pointed-P2 transcriptional activity by blocking its MAPK docking site

During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of a mitogen activated protein tyrosine kinase, called Rolled. Key nuclear targets of Rolled are two antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2), an activator. A critical regulator of this process, Mae, can interact with both Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the mechanism of Pnt-P2 regulation by Mae remained undefined. We find that efficient phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2 activity. Docking site blocking provides a new mechanism whereby the cell can precisely modulate kinase signaling at specific targets, providing another layer of regulation beyond the more global changes effected by alterations in the activity of the kinase itself.

Plasminogen activation by recombinant tissue plasminogen activator and potentiation by fibrin: a refined kinetic analysis

From previous studies, it is generally accepted that fibrin potentiates the tissue plasminogen activator (t-PA) mediated activation of plasminogen (PLgN) by substantially lowering the Km of the reaction. However, in the majority of these studies, kinetic constants for PLgN activation by t-PA were derived from experiments where dissimilar PLgN concentration ranges were employed in enzyme assays with and without fibrin. From this study, and employing a similar PLgN substrate range (0.07–1.0μM) in reactions with and without fibrin I (des-AA-), we show that by Michaelis-Menten kinetics, there is little alteration in the Km of the above reaction with fibrin addition, while kcat increases up to 10-fold. In the absence of fibrin I, recombinant two chain t-PA (rTCt-PA) yielded the following kinetic parameters: kcat 0.065s−1 and 0.30s−1, for Glu- and Lys-PLgN respectively, Km 0.45 μM for both. In the presence of fibrin I between 0.05 and 0.3μM, and with rTCt-PA as the enzyme, the activation of Glu- and Lys-PLgN obeyed Michaelis-Menten kinetics over the substrate range 0.07 to 0.7 μM. For PLgN activation at infinite fibrin concentration, values for kcat were estimated as 0.60s−1 and 1.25s−1, values for Km as 0.31 and 0.16 μM, and values for KF (the dissociation constant for the fibrin-rTCt-PA complex) as 0.075 and 0.070 μM, for Glu- and Lys-PLgN respectively. To determine whether the apparent Michaelis-Menten parameters for t-PA activation of PLgN are dependent on the employed substrate concentration range, the kinetics of this reaction were reassessed over a broad PLgN range (0.002-30μM). For rTCt-PA activation of Lys-PLgN, reaction kinetics were found to be non-standard, and consistent with cooperativity. The reaction yielded two sets of parameters by Michaelis-Menten kinetics; kcat values were 0.26 and 0.89s−1, and Km values were 0.30 and 2.7 μM. Taken as a whole, data presented here suggest that in the majority of previous studies, the kinetic constants estimated for reactions in the absence and presence of fibrin might be inappropriate for comparison, owing to the possibility of t-PA exhibiting non-standard Michaelis-Menten kinetics.

Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.

KINETICS OF ENZYMATIC PHOTOREACTIVATION STUDIED WITH PHAGE T1 AND ESCHERZCHZA COLI Bs‐1*

Abstract— The kinetics of enzymatic photoreactivation (PR) of u.v.‐induced killing was compared among E. coli Bs‐1, phage T1 in Bs‐1 and phage T1 in irradiated Bs‐1. The PR action spectrum showed no substantial difference between PR of Bs‐1 and PR of T1 in Bs‐1. The PR D37 (i.e. the PR dose required to reactivate all but 37 per cent of the reactivable lethal lesions) was found to decrease linearly with decreasing U.V. dose whether U.V. was given to produce pyrimidine dimers in Bs‐1 DNA, which then compete with irradiated T1 DNA for PR enzyme, or to Bs‐1 or T1 DNA to produce dimers serving as substrate for the PR enzyme. A generalized Michaelis‐Menten formula was used to analyze the data and the following conclusions were drawn. (1) The number of PR enzyme molecules per cell available for PR of T1 DNA inside the Bs‐1 host is only a quarter of the number available for PR of the Bs‐1 host itself. (2) The Michaelis constant Km for reaction of host‐DNA‐damage and PR‐enzyme becomes larger when the host damage acts as competitive inhibitor to PR of T1 DNA than when it is the substrate for PR enzyme. (3) PR enzyme retains almost all its initial catalytic efficiency even after about two‐hundred rounds of catalytic functioning. Conclusions (1) and (2) suggest that PR enzyme is concentrated within the nuclear area surrounding the host DNA.

The Deoxyribonucleic Acid Nucleotidyltransferase Activities of the Shope Fibroma: Partial Purification and Kinetics

Abstract DNA nucleotidyltransferase was partially purified from Shope fibroma tissue. The final preparation was free of terminal transferase but contained traces of deoxyribonuclease and nonspecific phosphodiesterase. The preparation was active with either native or denatured DNA as template. When assayed with native DNA as template, the activity was purified 61-fold, whereas when denatured DNA was used as template, purification was 179-fold. The Km for deoxyribonucleoside triphosphates for the native DNA- and denatured DNA-primed activities were 0.046 mm and 0.16 mm, respectively. The Km for native DNA varied with the source of the DNA. The denatured DNA reaction alone was inhibited by high concentrations of substrate. The Km values (20 to 25 µg per ml) for denatured DNA were almost the same for denatured DNA from four different sources, but the K's values varied widely (90 to 650 µg per ml). Brief treatment with DNase I greatly increased the Vmax of the native but not the heat-denatured DNA-primed reaction. The Km of the native DNA-primed reaction was not affected. Despite the affinities for template demonstrable kinetically, the DNA nucleotidyltransferases and DNA failed to form complexes that were stable in neutral sucrose gradients. The data for the reaction primed by denatured DNA suggest competition by 2 molecules of denatured DNA for a secondary binding site for template DNA. The increase in Vmax but not in Km of the reaction primed by native DNA after partial digestion of the template by DNase I may have been due to the increased ease of unwinding of the molecule after the introduction of single strand breaks, whereas the failure of the same preparations to exhibit enhanced priming activity after denaturation indicates the necessity for DNA of some minimum length for the denatured DNA-primed enzyme activity. The data suggest that the preparation contains two different DNA nucleotidyl-transferases, but the possibility that a single molecule is responsible for both activities is not ruled out.