The dynamic and thermodynamic properties of the complexes formed between anhydrotrypsin and the pancreatic trypsin inhibitors (Kunitz and Kazal) have been studied at different pH. They have shown that dehydration of Ser^s in the active site of trypsin hardly affects the binding properties of the enzyme to inhibitors. For example, at pH 8.0, 25°, the dissociation constant of the anhydrotrypsin-Kunitz inhibitor complex is 1.1 X 10~13 M as compared to 6.0 X 10-14 M for the trypsin-Kunitz inhibitor complex, and the dissociation constant of the anhydrotrypsin-Kazal inhibitor complex is 3.4 X -10 M as compared to 3.3 X 10~u M for the trypsin-Kazal -Because of their interest as models of heterologous proteinprotein interactions, associations of trypsin with protein trypsin inhibitors have been the object of extensive investigations during the last years both from the part of biochemists and from that of crystallographers. Two systems have been particularly well studied: the association of trypsin with soybean trypsin inhibitor (for references see Finkenstadt et al., , Sweet et al., 1974 and the association of trypsin with the pancreatic trypsin inhibitor1 discovered by Kunitz (for references see Lazdunski et al., 1974; Riihlmann et al., 1974).The three-dimensional structure of the basic pancreatic inhibitor (Kunitz) is now known with a resolution of 1.5 Á (Deisenhofer and Steigemann, 1974); that of the trypsin-PTI complex is known with a resolution of 2.8 Á (Riihlmann et al., 1973). This complex is extremely stable; its dissociation constant is 6 X 10"14 M and the half-life measured for its dissociation is about 17 weeks at pH 8.0, 25°( Vincent and Lazdunski, 1972).X-Ray analysis of the crystalline trypsin-PTI complex has shown that assembly of the partners was essentially stabilized by (i) 7 hydrogen bonds and about 200 van der Waals contacts, (ii) the formation of a salt bridge between the é-ammonium of Lysis in PTI and the /3-carboxylate of Aspn?, the essential residue in the specificity site of the enzyme, (iii) the formation of a covalent bond which is part of a tetrahedral adduct between the -OH function of Seri83, one of the essential residues in the
Specific acylation of the histidine residues of yeast hexokinase with diethylpyrocarbonate at pH 6.1 or 7.5 leads to a partial and reversible inactivation of the enzyme.The carboethoxylation of the histidine residues at pH 7.5 was faster than at 6.1 and the loss in activity was also higher.However the difference in the amount of inactivation was related to a greater instability of the carboethoxylated enzyme at pH 7.5.When the carboethoxylation was carried out at pH 7.5 and at 0 "C, only the histidines residues were modified (nine residues per enzyme subunit) and still 4001, of the activity was left.D-Glucose protects the enzyme activity in the course of the carboethoxylation at pH 7.5, but has no effect a t pH 6.1. However the rate and number of histidines acylated were the same as when the experiments were carried in absence of the substrate.All these results clearly indicate that the histidine residues are not implicated either in the catalytic or in the substrate binding site of yeast hexokinase.This was also confirmed by showing that the 600/, inactivated enzyme had the same K , as the native enzyme for both substrates D-glucose and Mg * ATP. Only v was affected.The inactivation effect of the carboethoxylation was not related to a displacement of the pH optimum of the activity, since the enzymes species having five and eight histidine residues modifiedrespectively in absence and presence of D-glucose at pH 7.5 gave the same pH profile of activity as the native enzyme.Absorption spectroscopy, fluorescence and ultracentrifugation studies have shown that the loss of enzymic activity in the course of the modification of the histidine residues is only related to changes of the protein conformation.Numerous investigations have been carried out on the mode of action of yeast hexokinase. I n contrast, the nature of the amino acid residues participating in the catalytic process are still unknown.I n a previous work [l], we have observed that yeast hexokinase is inactivated by photooxidation in presence of methylene blue. In the course ofthe photodynamic process, the loss of enzyme activity occurs by a biphasic process. At the initial rate of photooxidation one histidyl residues is modified with concommitant loss of 50 of the activity and change of the tertiary structure of the protein. I n the second phase of the photooxidation, multiple chemical effects were observed (oxidation of another 5 histidyl, 2 cysteinyl, 2 tryptophyl and 10 methionyl residues) which rendered the interpretation of the mechanism of photoinactivation more hazardous. Eur. J. Biochem. 39 (1973) Histidine groups have been described to be involved in the catalysis of several phosphoryl-transfer enzymes [2-41. From the photooxidation studies of hexokinase, we were unable to include or exclude definitively the role of this residue in the mechanism of action of the enzyme.Diethylpyrocarbonate has been shown, by several authors, to be a specific acylating agent of the histidine residues in proteins [4-91.I n this report the effect of this reagent ...
"Cord factors" are diesters of a,a-trehalose produced by Mycobacteriae and Corynebacteriae. Saponification of the "cord factor" of Corynebacterium diphtheriae gives an equimolecular mixture of homologues of the saturated corynomycolic and of the unsaturated corynomycolenic acids (I and 11, R = H). Mass spectrometry of their methyl esters confirms their structures (see Fig. 1 ,The peracetylated cord factor (IV, a, b, c) gave very weak molecular ions at m/e 1630, 1632 and 1634 showing apparently that it is a mixture of the diesters having respectively two saturated, one saturated and one unsaturated, and two unsaturated acyl radicals. Strong peaks a t mle 809 and 807 are attributed to the oxonium ions (V a and V b).These results as well as chemical degradations show that the "cord factor" of C. diphtheriae is a mixture of glycolipids (I11 a, b, c). La permethylation du glycolipide, ainsi que l'identification du tri-0-methyl glucose (effectukes par M. Yamakawa) ont Bt6 faites essentiellement selon No11 et coll. [S]. AcktylationEnviron 3 m g de substance, en solution dans 0,5ml de pyridine sont gard6s pendant une nuit apres addition de 0,5 ml d'anhydride ac6tique. Aprks Bvaporation B sec sous vide, environ 0,1 mg du residu a Bt6 utilise directement pour la spectrometric de masse. XpectromCtrie de rnasse
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