1. The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the -SH group ofthe enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in aHaO it is concluded that a mercaptideimidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.2. Iodoacetamide reacts with the -SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo-, chloro-, and fluoroacetamide. This might be due to the less polar environment around the -SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo-and chloroacetamide in the presence of dioxane.3. By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH z 9, whereas the overall protein structure is stable up to pH zz 11. D-2-Bromo-n-butyramide reacts with thiolsubtilisin about 60 times as fast as D-2-bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala-152 and Thr-220 of thiolsubtilisin may form van der Waals interactions with D-2-bromon-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin.Thiolsubtilisin, an -SH analog of the serine protease subtilbin, can be produced by the replacement of the reactive serine residue at the active site by a cysteine residue [l]. Thiolsubtilisin can be regarded as a model of thiol proteases, such as papain, inasmuch as both thiolsubtilisin [2] and papain [3] have an imidazole ring in the immediate vicinity of the thiol group, as clearly indicated by X-ray diffraction studies. However, thiolsubtilisin is not a protease, it can only catalyze the hydrolysis of active esters [a].In a recent paper we have pointed out that the catalytic action of papain depends on the formation of a mercaptide-imidazolium ion-pair at the active site [5]. On the basis of acylation of thiolsubtilisin, we have also suggested that the reactivity of the thiol group is highly affected by an interaction with the The detailed study of alkylation of Carlsberg thiolsubtilisin is presented in this paper. Unlike acylation, alkylation is a simple nucleophilic displacement reaction, not complicated by the formation of an intermediate, therefore the study of alkylation, rather than of acylation, offers more conclusive evidence for the chemical behavior of the thiol group. Carlsberg thiolsubtilisin was preferred to the Novo enzyme because of its higher stability. We have found that around neutral pH values there is indeed an interaction between the thiol and imi...

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Die Umsetzung des Triethylammoniumsalzes von CAMP (I) mit Ethylenoxid (II) ergibt die Ammoniumverbindung (III) und nicht ,den Phosphorsäureester (IV).