Jean-Jacques Béchet

Mise en évidence d'un effet allostérique lors de l'hydrolyse du p-toluéne sulfonyl--arginyl-méthyl ester par la trypsine

Abstract Some evidence for an allosteric effect during tryptic hydrolysis of p- toluenesulfonyl- L methyl ester Kinetic and thermodynamic study of trypsin (EC 3.4.4.) hydrolysis of p- toluene-sulfonyl- L -arginine methyl ester has shown as activation by high concentrations of the substrate. The analysis of the results obtained at different pH and temperature values suggest that there are two different substrate-binding sites on the enzyme molecule: a principal site an auxiliary one. The binding of a substrate molecule on this last site, probably induces a discrete and reversible change in the configuration of the enzyme, so that the catalytic site takes an optimal conformation. The second substrate molecule reacts as an allosteric effector; its binding on the trypsin molecule increases the catalytical activity of the enzyme.

Effet du pH sur la fixation d'inhibiteurs compétitifs synthétiques par la trypsine☆

Abstract Conformational changes in trypsin (EC 3.4.4.4) induced by the binding of competitive inhibitors (benzylamine, butylamine, N-α- benzoyl- l -arginine ) and, in some favourable cases, of substrates ( N-α- benzoyl- l -arginine ethyl ester, N-α- benzoyl- l -arginine amide) have been studied at 10°, in the pH range from 1.5 to 12.5. The formation of the trypsin-inhibitor (or trypsin-substrate) complex produces changes in the enzymes rotatory power; this modification is small at neutral pH but substantial in acid and alkaline pH ranges where trypsin is reversibly denatured. It seems that a more compact structure of the protein results from the formation of the complex. It has been shown by spectrophotometric measurements in alkaline medium that this conformational change is accompanied by the burying of one tyrosine residue which ionizes normally in the free enzyme. The formation of the enzyme-substrate complex coincides with a proton release at pH values lower than 6. Results obtained both by proton titration at pH 4.1 and by polarimetric measurement of the affinity of benzylamine for trypsin at pH 3.5 and 2.5 indicate the existence of an interaction between the positive charge of the inhibitor and the negative charge of a carboxyl group of the enzyme having a pK of 4.7. The role of the substrate in maintaining the structural and catalytic properties of trypsin is discussed.

Disuccinimidyl esters as bifunctional crosslinking reagents for proteins: Assays with myosin

Bifunctional crosslinking reagents have been used in studies of the spatial arrangement of muscle contractile proteins, such as myosin and actin, either in their soluble forms [ 1,2], or in synthetic filaments [3-51, or even in myofibrils [3]. The most commonly used reagents are the bisimidates, which are very reactive, but also quite unstable in aqueous solution; incomplete substitution and unexpected side reactions furthermore occur if the crosslinking reaction is at pH -8 [6,7]. A disuccinimidyl ester, the dithiobis (succinimidyl propionate), DSP, that does not have these drawbacks and contains, moreover, an easily cleavable disulfide bond, has been described [8]; unlike the bis-imidates, this reagent allowed the crosslinking of the two heads of a myosin molecule [ 21. Owing to the high chemical reactivity of DSP and its stability in water, we thought it interesting to synthesize a series of disuccinimidyl esters of various chain lengths (table 1). These include non-cleavable reagents (compounds I-IV), and also reagents with either a uic-glycol (compounds V, VI) or an ethylenic bond (compound VII); the crosslinks formed by these last compounds can in principle be cleaved,

Inactivation of α-chymotrypsin by a bifunctional reagent, 2-bromomethyl-3,1-benzoxazin-4-one

Abstract α-Chymotrypsin is selectively and irreversibly inactivated by 2-bromomethyl-3, i -benzoxazin-4-one (Compd Ib) at neutral pH. Reaction of the enzyme with this activated ester leads rapidly to a relatively stable acyl-enzyme in which intramolecular alkylation of a single methionine residue (likely methionine-192) followed by hydrolysis of the acyl-enzyme bond occurs. Kinetic evidence for such a process is found in the measurements of proflavin displacement accompanying the reaction. The irreversibly modified enzyme still possesses its intact active site but its activity towards specific substrates is altered. Alkylation of the enzyme increases mainly K m but does not change appreciably k cat . The inhibition constants of specific inhibitors such as proflavin or indole are increased several times. From crystallographic data it is known that methionine-192 forms the lid of the specificity cavity in the enzyme. Therefore a bulky substituent such as the 2-acetamido benzoic acid on the sulfur atom of this methionine might sterically hinder the substrate binding by blocking the approach of the binding site.

Structural relationship of myosin isoenzymes: Proteolytic digestion patterns of heavy chain components from fast muscles, and comparison with other muscle types

Muscle myoslns are made up in all known cases of one pair of heavy chains and two pairs of light chains, the so-called alkali and regulatory subunits. Muscle type specificity of myosin has been ascribed to differences in the light and also the heavy subunits. indeed, the heavy chains of myosins from slow, fast, and cardiac muscles have been reported to differ immunologically [ 11, and the last two also chemically 12 3; these slow, fast and cardiac myosins are known to display different ATPase activities. Electrophore~s of myosin in its native state has demonstrated that in each of these muscles it is present in several isoenzymic forms [3,4]. In the case for instance of the fast-twitch muscles, such as chicken pectoralis and posterior latissimus dorsi or rabbit skeletal muscle, three myosin populations are found, which differ in their contents of the two alkali light chains, the subunits A, and Al. They contain, respectively, two Al, two AZ, and one of each light chain per molecule of myosin, and correspond therefore to Al and Aa homodimers and the AZ AZ heterodimer [4,5]. To determine whether the differences between these myosin components are confined to their alkali light chains, or extend also to the heavy chains, we have carried out a comparative study of the proteolytic digestion patterns of the latter from all the three fast muscle isoenzymes. After electrophoretic separation in non-dissociating conditions, the heavy chains of each resolved isoenzyme were separated from the light chains and recovered by way of a second electrophoresis

Étude de l'inhibition de l'activité estérasique de la trypsine par les dérivés acides et amides des esters spécifiques

Abstract The inhibition of the esterase activity of trypsin (EC 3.4.4.4) by acid and amide derivatives of specific ester substrates has been determined at several pH values between 5 and 9 and at 25°. With either specific or non-specific ester substrates, the inhibition by the acid derivatives is stronger at low pH. The reverse is the case for the amide derivatives. The sigmoid curve of pKi dependency versus pH for both series of inhibitors suggests a very close interaction between the —COX group of the acid or amide derivatives and the histidine at the active center of the enzyme during the formation of the first non-covalent complex. The affinities of these different specific derivatives for the enzyme have been compared at the optimal pH value of the enzymatic activity.

Binding of competitive inhibitors to the different pH-dependent forms of trypsin☆

Abstract Some of the rotatory, spectral, ionic and catalytic properties of trypsin (EC 3.4.4.4) have been studied, in the pH range from 1.5 to 12.5, in the presence and absence of synthetic competitive inhibitors. In the alkaline and acidic pH ranges where trypsin is reversibly denatured, the formation of the trypsin-inhibitor complex protects the enzyme against transconformation; at neutral pH, where trypsin is in the native form, the complex formation sometimes induces a change in the enzyme structure. In the alkaline pH range (a) the rotatory dispersion curve of the complex is pH independent; (b) the formation of the complex induces a decrease of the enzyme absorbance; (c) the formation of the complex produces a proton uptake by the enzyme; (d) the affinity of inhibitors decreases when the pH increases. All these results can be related to the ionization of an enzyme group having an apparent pK of about 10. In the acidic pH range (a) the rotatory power of the complex is pH independent, and (b) the complex formation produces a proton release by the enzyme. The changes in the ionic and rotatory properties of trypsin as a function of inhibitor concentration have allowed the evaluation of the dissociation constant of the benzamidine-trypsin complex between pH 4.5 and 1.5. A scheme for the interaction between the inhibitor and the different pH dependent forms of trypsin is proposed: it accounts for the pH dependence of the complex dissociation constant and also for the dependence of the number of protons released by the enzyme. The lowering of the inhibitor affinity in the acidic pH range is due to the protonation of two enzyme groups, one having an apparent pK of 3.7, the other a true pK of 4.5. At neutral pH, the enzyme-inhibitor complex formation may induce a change in both the enzyme and the inhibitor structure; formation of the trypsin-proflavin complex, for instance, makes the inhibitor optically active.

Separation of polar, steric and specific effects in the α-chymotrypsin-catalyzed hydrolysis of acyl-substituted p-nitrophenyl esters

Abstract The α-chymotrypsin-catalyzed hydrolysis of various acyl-substituted p-nitrophenyl esters has been studied at 25°C, between pH 6 and 8. The substituent effects on the deacylation rates of corresponding acyl-chymotrypsins have been analyzed using the Taft-Ingold relationship and contributions of polar, steric and specific effects, separated. A specificity constant S has been defined and its value discussed in relation to the structure of the substrate.

Correlation among the proton charges and molecular masses of myosin subunits

The molecular masses and isoelectric points of myosin light and heavy chains were calculated from their known primary sequences and their respective distribution in a two‐dimensional graph is displayed. Implications for the electrophoretic study of myosin subunits are inferred from this analysis.

The influence of temperature on the α-chymotrypsin-catalyzed hydrolysis of N-acylamino acid p-nitrophenyl esters*

Summary The effect of temperature on the deacylation step in the α-chymotrypsin-catalyzed hydrolysis of several N-acyl-L (or D)- amino acid p-nitrophenyl esters has been studied. A compensation in the activation parameters ΔH ‡ and ΔS ‡ for this step has been found. This phenomenon is discussed in terms of the breaking or formation of specific interactions between the substrate and the enzyme, in the transition state of the deacylation reaction.