Date of Award

2001

Degree Name

Chemistry

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

William D. Price

Abstract

On-line reaction monitoring using electrospray ionization mass spectrometry was investigated with the goal of kinetically distinguishing the isomeric amino acids leucine and isoleucine. Leucine and isoleucine are isomers of identical mass (113.0946 Da) which are impossible to distinguish by their mass-to-charge ratios. These isomers are a source of ambiguity when sequencing peptides with exoproteases and mass spectrometry. Here we investigated the kinetics of on-line enzymatic reactions involving exoproteases for differentiating between leucine and isoleucine.

The kinetics of the stepwise exopeptidase degradation of 12 leucine/isoleucine isomeric peptides was monitored in real-time with a Finnigan Mat LCQ with an electrospray ionization source. Data was analyzed within the Michaelis-Menten formalism via ion intensity-time curves and Lineweaver-Burke plots. A difference in the kinetic values for the two isomeric amino acids was expected due to the highly specific nature of enzymes. Based on the observed data leucine is hydrolyzed 2-3 times faster than isoleucine. The Michaelis constant, Km, was also larger for leucine than isoleucine by a factor of four.

Subject(s)

Leucine.

Proteolytic enzymes.

Enzyme kinetics.

Chemistry, Physical and theoretical.

Biochemistry.

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