Phenothiazine-N-carbonyl chloride, a specific inactivator of chymotrypsin

Abstract

Phenothiazine-N-carbonyl chloride inactivated chymotrypsin and trypsin by means of a 1:1 stoicheiometric reaction. Its reaction with chymotrypsin was 29 times as fast as that with trypsin and was inhibited by indole. The reaction of phenothiazine-N-carbonyl chloride with chymotrypsin resembled an enzyme-substrate reaction in which the deacylation step is rate-limiting. Slow deacylation occurred, resulting in complete regeneration of active enzyme in 15h. The pH-rate profile of the inactivation process had a maximum at pH7.8. These data and other evidence indicate that the reaction of phenothiazine-N-carbonyl chloride with chymotrypsin exhibits ;kinetic specificity'. Therefore any hypothesis that attempts to describe the topography of the active site of chymotrypsin should take into account the reactivity of phenothiazine-N-carbonyl chloride. The above findings, as well as recent reports of others, are examined within the context of a hypothesis given in an earlier paper (Erlanger, 1967).

Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation

“…For instance, p-bromophenacyls and iodoacetamides have been found to selectively alkylate carboxylic acid moieties of pepsin and ribonuclease T1 respectively. [582][583][584] However, the applicability of these reagents is not general and is appropriate on specific substrates only.…”

Chemical modification of histidine and lysine residues of crotoxin

“…Briefly stated, this thesis is that there exist no specific protein reagents, but only specific protein reactions. p-Bromophenacyl bromide had been shown specifically to esterify one aspartyl group in pepsin [19,20] long before it was found specifically to alkylate a histidine residue in the active site of phospholipase A2 molecules from widely different sources [ l-5, 21,22]. We now report that the active site of crotoxin contains a histidine which is not accessible to that reagent until this protein complex is dissociated into its two components.…”

The role of carboxylic acid groups in the action of glucoamylase I

“…A variety of methods have been used for the chemical modification of carboxyl groups in proteins and these can be valuable in structure-function correlations. For example, p-bromophenacyl bromide [6] and l-diazo-6-phenyl-3-toluenesulphonamidobutan-2-one…”