I. The Kinetics of the Alpha-Chymotrypsin Catalysed Hydrolysis of Acetyl-L-Tyrosinhydroxamide. II. Genetic Factors Influencing the Activity of Tryptophane Desmolase in Neurospora crassa

Citation

Hogness, David Swenson (1953) I. The Kinetics of the Alpha-Chymotrypsin Catalysed Hydrolysis of Acetyl-L-Tyrosinhydroxamide. II. Genetic Factors Influencing the Activity of Tryptophane Desmolase in Neurospora crassa. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4Y9X-NZ51. https://resolver.caltech.edu/CaltechETD:etd-04232003-104814

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. From an analysis of the pH-activity curves of the systems alpha-chymotrypsin-acetyl-L-tyrosinamide and alpha-chymotrypsin-acetyl-L-tyrosinehydroxamide it has been concluded that in the latter system the active specific substrate is acetyl-L-tyrosinehydroxamide and not acetyl-L-tyrosinehydroxamate ion. At pH 7.6 and 25[degrees]C, in aqueous solutions 0.3 M with respect to the amine component of a tris-(hydroxy-methyl)-aminomethane-hydrochloric acid buffer, the kinetics of the alpha-chymotrypsin catalysed hydrolysis of acetyl-L-tyrosinehydroxamide have been found to be similar to those observed previously for this enzyme and specific substrates of the acylated alpha-amino acid type at pH 7.9 [plus or minus] 0.1 and 2.5[degrees]C in aqueous solutions 0.02 M with respect to the amine component of the same buffer system. Acetyl-D-tyrosine ethyl ester, acetyl-D-tyrosine hydrazide, acetyl-D-tyrosinehydroxamide and acetyl-D-tyrosinamide were found to be competitive inhibitors of the alpha-chymotrypsin catalysed hydrolysis of acetyl-L-tyrosinehydroxamide at pH 7.6 and 25[degrees]C, under the conditions previously specified, and from these and other data it was concluded that proteins, amides, esters, hydrazides and hydroxamides are hydrolysed at the same single active site on the enzyme. Preliminary to an examination of the effect of genetic factors on tryptophane desmolase activity in Neurospora, this enzyme was extracted from a reference strain and partially purified. The kinetics of this tryptophane desmolase system was investigated and the apparent K[subscript m] values of indole, L-serine and pyridoxal phosphate determined. No tryptophane desmolase activity could be found in a tryptophaneless mutant, C83. The tryptophane requirement of this mutant and the lack of tryptophane desmolase activity were found to be associated with the same single gene. The reversion of C83 to wild type under the influence of ultraviolet irradiation was studied, and it was shown that the C83 mutant can backmutate. The backmutant was found to contain a tryptophane desmolase system which could not be distinguished from the tryptophane desmolase from wild type on the basis of the kinetic criteria that were applied. A histidineless mutant, C84, was found to exert a control on the tryptophane desmolase activity of Neurospora. From a limited amount of evidence it was tentatively concluded that the histidine requirement and the alteration of the tryptophane desmolase activity are the effects of the same single gene in the C84 mutant.

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