Mechanism Of Enzyme Action Research Articles

On the mechanism of enzyme action. LVII. Interaction between trypsin and ovomucoid

Trypsin, ovomucoid, and their equimolecular complex have sedimentation constants of 2.5, 2.4, and 3.7 – 3.9 S, respectively. While trypsin can bind only one ovomucoid molecule, ultracentrifugal and electrophoretic data show that more than one molecule of trypsin can react with each molecule of ovomucoid. The dissociation constant of the equimolecular complex is 5.8 × 10 −9 M and of the complex involving two trypsin and one ovomucoid molecule is about 2.5 × 10 −6 M, as dedetermined by activity measurements. The isoelectric points of the two proteins and their equimolecular complex are at about pH's 10.8, 4.3, and 9, respectively. The complex behaves on electrophoretic examination as an independent protein, stable over an extended pH range, and dissociating only around pH 3.8. The trimolecular complex has a mobility intermediate between that of the equimolecular complex and free trypsin. It has a more limited pH range of stability, and dissociates readily around pH 6. The presence of either of the complexes does not cause a change in the electrophoretic mobilities of the other components present. The inhibition of trypsin by ovomucoid is considered to be another instance of competitive substrate inhibition.

On the mechanism of enzyme action. LVIII. Acetyltrypsin, a stable trypsin derivative

Acetylated trypsin is homogeneous in the ultracentrifuge and presents a slight inhomogeneity in electrophoretic migration. Its isoelectric point is about pH 3.8. Owing to blocking of the ϵ-amino groups of lysine, acetylated trypsin is stable in alkaline media and is not subject to self-digestion. Its stability in acid media is, however, smaller than that of the unmodified enzyme. Its thermal stability is also slightly lower than that of trypsin. Ca ++ ions stabilize both enzymes. The pH optimum of digestion for casein is shifted to more alkaline pH values when the enzyme is acetylated. The temperature optimum is slightly lower. The Michaelis-Menten constant for trypsin and acetyltrypsin with casein is K m = 4.4 × 10 −6 M and K m = 3 × 10 −5 M, respectively. Acetyltrypsin combines also with ovomucoid and is inhibited by it, though to a much lesser degree than the unmodified enzyme. The dissociation constant of the equimolecular acetyltrypsin-ovomucoid is 1.3 × 10 −6 M using hemoglobin as substrate, and 3.5 × 11 −7 M with casein. The inhibition seems therefore to be competitive.

The Mechanism of Enzyme Action

The Mechanism of Enzyme Action

The Mechanism of Enzyme Action

The Mechanism of Enzyme Action

The Mechanism of Enzyme Action

The Mechanism of Enzyme Action

On the mechanism of enzyme action. LV. A study of the interaction between calcium and trypsin

1. 1. In the neighbourhood of pH 5 the electrophoretic analysis of crystalline trypsin results in patterns characteristic of a homogeneous protein, while the addition of calcium, manganese or calcium ions (0.033 M) causes the moving boundary to separate into two distinct peaks. The two components of trypsin have comparable stability both in the presence or absence of calcium ions and, if separated electrophoretically, possess similar proteolytic activity. 2. 2. Trypsin has a sedimentation constant of 2.4–2.5 S at the pH values of 2.3 and 8. On the acid side of pH 2.3, the denaturation causes a slow appearance of faster sedimenting components, whereas at pH 8, the self-digestion prompts the appearance of slower sedimenting breakdown products. The rate of sedimentation of the remaining unaltered trypsin appears unaffected by the presence of either the faster or the slower sedimenting components. 3. 3. In the intermediate pH range, around pH 5, the sedimentation constant may increase up to 3.5 S, depending upon the conditions, while the enzyme sediments as a homogeneous protein. This is due to a reversible aggregation equilibrium which is strongly temperature dependent. Calcium prevents the aggregation. 4. 4. Trypsin has to be viewed as a heterogeneous protein consisting of two components. In the absence of calcium, manganese or cadmium, in the neighbourhood of pH 5, the reversible molecular interaction of the two components induces the aggregation and results in apparently homogeneous electrophoretic and ultracentrifugal patterns. The addition of calcium or of the other two ions prevents the interaction, restores the normal sedimentation behaviour, and reveals the true electrophoretic heterogeneity of the enzyme.

On the mechanism of enzyme action. LIII. The amphoteric properties of trypsin

Several methods were employed for the preparation of salt-free trypsin samples which were used to determine the electrometric titration curves of the enzyme. These curves point to a maximum acid-binding capacity below pH 2. Stoichiometric analysis indicates the presence of 6 carboxyl groups per 10,000 g. of proteins, 1 imidazole group, and 13 hydroxyl-binding groups. Calcium has a specific effect on the titration curves by increasing the acidity of the carboxyl groups in the pH range 3.5 – 5. This effect is not shown by potassium, sodium, or even the bivalent magnesium ion. It is attributed to the formation of a specific complex between the enzyme and the calcium ions, involving the carboxyl groups of the protein. The equally specific protective effect of calcium on the self-digestion of trypsin can therefore be explained by assuming the formation of a complex which stabilizes the enzyme. There is good qualitative agreement between the pH dependence of the electrophoretic mobility of the enzyme and the titration curves. In addition to stabilizing the enzyme, calcium also slightly increases its activity. The investigation of the dependence of the proteolytic activity of trypsin on the pH in the presence or absence of calcium at various temperatures indicates that both effects of calcium ions, the protective effect and the increase in activity, are due to a shift of the equilibrium between native and denatured enzyme in favor of the active form.

On the mechanism of enzyme action. LII. The usefulness of 1-C 14 acetate for the study of carbohydrate → fat synthesis in Fusarium lini Bolley

1. 1. When acetate and glucose are utilized as a mixed carbon source, a greater percentage of the carbon present is converted into fat and sterol in the presence of relatively higher concentrations of acetate. 2. 2. From the data presented it may be concluded that the major pathway for the formation of unsaturated fatty acids in Fusarium lini Bolley is via a dehydrogenation mechanism. 3. 3. Carbohydrate yields C 2 units which are utilized for the formation of saturated fatty acids. 4. 4. The results indicate the preferential utilization of acetate carbon for the synthesis of both fat and sterol and that the majority of the carbon atoms needed for the formation of mycelium was derived from glucose.

On the mechanism of enzyme action. LI. Effects of naphthoquinones on carbohydrates → fat conversion in Fusarium lini Bolley

The effects of naphthoquinones on certain phases of growth and fat formation in Fusarium lini Bolley were studied. The addition of the effectors resulted in lowered mat weights, fat coefficients, sterol concentrations, and increases in the unsaturation of the fatty acids formed. Extension of this latter observation showed increased oleic and linoleic acid contents. Pyruvic acid accumulation increased with increasing amounts of added compounds, and was much greater than in the medium of the control, in some cases, as high as ten times the amount in the control. The conclusion arrived at is that quinones exercise two main functions in their participation in growth processes and fat formation in Fusarium lini Bolley.

On the mechanism of enzyme action. XLVIII. Investigation of the fat of Fusarium lini Bolley by means of urea adducts

The usefulness of urea adducts for the fractionation of natural fatty acids has been demonstrated. Stearic acid has been isolated and identified from the fat of Fusarium lini Bolley by purification of the solid fraction obtained from the urea adduct of its fatty acids. The conversion of stearic acid to fatty material with a higher iodine value has provided further evidence for the enzymatic formation of unsaturated fatty acids in Fusarium lini Bolley by means of the action of a fatty acid dehydrogenase system.

On the mechanism of enzyme action. XLIX. The effect of acetate on fat formation in Fusarium lini Bolley and the influence of added naphthoquinones

1. 1. When Fusarium lini Bolley (FlB) is grown on an acetate-containing medium the fat formed is more saturated than the fat formed when glucose is employed as substrate. These differences were shown to be due to the higher oleic acid content in the fat formed by Fusarium lini Bolley from glucose. These differences are thought to be due to the fact that when glucose is used as the carbon source, alcoholic fermentation is proceeding concurrently with fat formation. 2. 2. Acetate as a substrate for fat formation is about 43% more advantageously utilized than glucose in the case of Fusarium lini Bolley. 3. 3. The mat weights and the amounts of fat formed increase linearly with the concentration of acetate employed, up to a value of 2%. 4. 4. When various naphthoquinones were added to the culture media containing acetate as the carbon source, the mat weights were not appreciably lowered when compared to the control. The data indicate that, in general, these quinones have very little effect on a system utilizing acetate as the carbon source.

On the mechanism of enzyme action. L. The influence of naphthoquinone on the mechanism of fat formation in Fusarium lycopersici

Fusarium lycopersici was continually subcultured giving rise to wide variance in the quantities of lycopersin formed by this organism. Two cultures were selected (one with about 25 mg. lycopersin/g. mycelium and the other with about 1 3 of this amount) in experiments in which different quantities of naphthoquinone were added to the media prior to inoculation. There was noted a relationship of mat weights, fat coefficients, iodine values of fatty acids, and composition of the fats formed with lycopersin concentrations. These findings point to an interaction of lycopersin with the dehydrogenase systems operative in fat formation and, also, to the distinct-action of this natural pigment in the enzymatic reactions of its host.

Mechanism of enzyme action.

Mechanism of enzyme action.

On the mechanism of enzyme action. XLVII. Effects of high intensity electron bombardment on crystalline trypsin

Summary The effect of ionizing radiations on enzymes in solution is much more complex than on enzymes in the dry state. The radiation dose required to cause a certain amount of inactivation of the enzyme will be as much as 170 times higher in the dry state, than in some dilute solutions. Any intermediate values can be obtained by changing the conditions of the irradiation of the enzymes in solution. The factors influencing the socalled “solvent” effect were found to be: concentration and nature of the buffer solution, pH, and temperature. The inactivating effect of electron bombardment on trypsin is considerably enhanced by increasing the acidity of the enzyme solution. This is in contrast with the maximum stability of this enzyme at pH 2.3 when not exposed to radiation. Calcium ions have no specific effect on the inactivation of trypsin caused by electrons, although, under normal conditions, these ions stabilize the enzyme. It can be concluded, therefore, that factors which contribute toward an increased stability of an enzyme under normal conditions have no correlation with the effects of ionizing radiation on the enzyme.

On the mechanism of enzyme action. XLVI. The effect of certain ions on crystalline trypsin and reinvestigation of its isoelectric point

Calcium and manganese ions are effective protectors of crystalline trypsin in alkaline solutions. Other ions seem to have no significant influence. The effect of calcium is due to a decrease in the rate of autodigestion of trypsin but does not affect the rate of tryptic digestion of other proteins. The isoelectric point of trypsin is in the neighborhood of pH 11, as determined by electrophoresis analyses. This high isoelectric point is correlated with the amino acid composition of trypsin and its amide nitrogen content.

On the mechanism of enzyme action. XLV. The role of certain dicarboxylic acids in the formation of oxalic acid by wood-destroying molds

Alkalinity of the medium was shown to be the chief factor involved in the accumulation of oxalate by T. cinnabarina. Glutamate and aspartate are shown to lead to oxalate with this organism and with L. lepideus by dehydrogenation to α-ketoglutarate and oxaloacetate, respectively. Malate was also shown to be dehydrogenated. It is proposed that oxaloacetate may either undergo β-decarboxylation to yield CO 2 and pyruvate, or splitting by coenzyme A to yield oxalate and acetylated coenzyme A. The reversal of this latter reaction is suggested as the explanation of the disappearance of oxalate from culture media. The reduction of resazurin by the dehydrogenase systems of the molds is inhibited by cyanide, indicating the participation of metal systems, such as the cytochromes.

On the mechanism of enzyme action. XLIV. Codetermination of resazurin and resorufin in enzymatic dehydrogenation experiments

A method has been evolved for the quantitative codetermination of resazurin and resorufin. Examples of its use with erythrocytes and with succinic dehydrogenase of rat liver are presented.

REVIEW OF BOOKS AND ARTICLES

Book reviewed in this article:Klein, Henry. Dental effects of accidentally fluorinated water: I. Dental caries experience in deciduous and permanent teeth of school age children.Klein, Henry. Dental effects of community waters accidentally fluorinated for nineteen years. II. Differences in the extent of caried reduction among the different types of permanent teeth.McKay, F. S. Mss control of caries through the use of domestic water supplies containing fluorine.Phillips, R. W., and Swartz, Marjorie L. Effect of fluorides on hardness of tooth enamel.Proceedings of the Michigan dental caries conference.McClure, F. J. Fluorine in dentin and enamel of sound and carious human teeth.Dreizen, Samuel, and Spies, T. S. Further studies on the association between the products of protein putrefaction and dental caries activity.Blayney, J. R. and Tucker, W. H. The Evanston dental caries study. II. Purpose and mechanism of the study.Rapp, G. W. The mechanism of enzyme action. Oral Surg., Oral Med., & Oral Pathology.

The mechanism of enzyme action

The mechanism of enzyme action

Cryolysis of Lyophilic Colloids, and Its Bearing on the Mechanism of Enzyme Action

Cryolysis of Lyophilic Colloids, and Its Bearing on the Mechanism of Enzyme Action