A Study of the Essential Groups of β-Amylase.¹ II. Sulfhydryl Groups

Abstract

Vol. 67 groups or botti are necessary to the amylase activity. The extreme slowness of this irreversible inactivation, however, favors the idea that tyrosine rather than amino groups are involved. This conclusion was strengthened by further study. When the log fraction of the amylase activity which remained after inactivation by nitrous acid and subsequent reactivation with hydrogen sulfide was plotted against time, the irreversible inactivation was found to be first order with respect to the amylase. Furthermore, the constant obtained for this reaction was of the same order of magnitude as that obtained under similar conditions by Little and Caldwell315 for the formation of the azo compound with pure tyrosine. In addition, the same constant was obtained when the inactivation and reactivations were repeated with another concentration of the amylase.These results appear to rule out any appreciable influence either of deaminazation or of drastic oxidations and lead to the conclusion that free tyrosine is essential to the activity of betaamylase.

Specific Group Reagents for Proteins.

“…If, under these conditions, the biological activity of a purified protein is first lost and then, after reversal of the reaction, regained, the group in question can be said to play a positive role in the biological activity. Such conditions have been met in only a limited number of cases (e.g., 148, 159, 168, 335, 336). More often investigators have used reagents of doubtful claim to specificity, based mainly on the analogy to their reactions with other proteins.…”

“…Some of the various disadvantages mentioned above have with nitrous acid is discussed in detail in Section III. It has been used by Philpot and Small (256) to study the essential nature of the tyrosine groups of pepsin, by Weill and Caldwell (336) with /3-amylase, and by others (table 2). Sizer (301) has claimed that the enzyme, tyrosinase, can oxidize a few tyrosine groups of some proteins, but his conclusions have been questioned (91).…”

“…Iodine has often been used as an oxidizing agent in the study of SH enzymes (cf. 31,157,158,223,336).…”

“…It is thus possible that groups that do not by themselves combine with formaldehyde, may be involved in toxoid formation through the secondary cross-linking reactions. ions trivalent arsenicals (19,297) as well as other mercury compounds (19,158,159,336) act similarly, although their effectiveness appears to vary (19,223).…”

Non-Oxidative Enzymes

“…149) believe that free sulfhy dryl groups are essential components of �-amylase of barley and bar ley malt. They base this conclusion on the results of studies of the inhibitory effects of various reagents on the activity of highly puri fied preparations of the enzyme.…”