Date of Award

Spring 5-2011

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry and Biochemistry

School

Mathematics and Natural Sciences

Committee Chair

Robert Bateman

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Douglas Masterson

Committee Member 3

Jeff Evans

Abstract

The reaction mechanisms employed during non-ribosomal peptide (amide) synthesis in the non-ribosomal modular enzyme complex and in the enzyme glutaminyl cyclase were also used for spontaneous peptide (amide) synthesis in a buffered, abiotic solution. Analytical analyses revealed that spontaneous peptide (amide) bond formation occurred under mild prebiotic conditions during both the thioester-based production of phenylalanine peptides and peptidyl thioesters, and the spontaneous cyclization of glutamine t-butyl ester (Gln-tbe) into pyroglutamic acid t-butyl ester. Internal standard-based quantitative MS determined that 4.9 x 10-5 sec-1 was the rate constant for spontaneous Gln-tbe cyclization in imidazole buffer, pH 7, at 50°C, and that the enzyme glutaminyl cyclase exhibited a rate enhancement of 1.0 x 106 over the non-enzymatic rate. Interestingly, the rate of Gln-tbe loss at neutral pH was significantly affected by t-butyl ester hydrolysis. The rate of amino acid t-butyl hydrolysis has not been reported; however, in this study the experimental rate of Asn-tbe was determined to be 1.5 x 10-5 sec-1 . Hydrolysis of a t-butyl ester group readily occurs under acidic conditions and the amino acid t-butyl ester hydrolysis at neutral pH could be a result of intramolecular hydrogen bonding. Arrhenius and Eyring plots determined the energy of activation, activation enthalpy, and activation entropy to be 9.5 x 10-4 J mor1, 9.5 x 10-4 J mor1, and -1.5 x 10-5 J K-1 mor1, respectively. Both cellular and prebiotic environments undergo constant changes in reaction conditions, and a detailed kinetic investigation revealed that the rate constant for Gln-tbe degradation was not affected by buffer type (imidazole, citrate), buffer concentration (0 to20 mM), or pH level (4 to 7). In addition, the reaction rate increased linearly as temperature was increased (30 to 60°C). The correlation between enzymatic and non-enzymatic peptide (amide) bond formation under mild prebiotic conditions presented the transamidation and thioester routes as possible methods for ancestral peptide (amide) bond formation.

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