Abstract

Enzyme IImtl of Escherichia coli was purified from mtlA-overexpressing E. coli strain LJ1112 extraction of isolated membranes with deoxycholate, followed by hexyl agarose and ω-amino hexyl agarose chromatography in Lubrol PX. A 10-fold increase in total pure protein and 2.5-fold increase in specific activity over previously reported procedures was obtained. Tryptic fragments of Enzyme IImtl were also purified and identified as the membrane and cytoplasmic domains by protein sequencing and SDS-PAGE. The fluorescence spectrum of Enzyme IImtl suggested that two populations of tryptophan residues existed with emission maxima at 322 and 344 nm, corresponding to buried and partially exposed tryptophans, respectively. Addition of mannitol to Enzyme IImtl resulted in a 20% increase in fluorescence with no observable shift in the emission maximum, as did substrates glucitol, perseitol and arabitol. KDs were estimated as 0.6 μM, 0.15 mM, 1.6μM and 0.5 mM for each of the substrates, respectively. Equilibrium dialysis gave a KD of 0.15 μM for mannitol. The membrane fragment, but not the cytoplasmic fragment, gave nearly identical fluorescence and equilibrium dialysis behavior for mannitol, showing that the mannitol binding site is localized to the membrane fragment. Inactivation of Enzyme IImtl with N-ethylmaleimide did not alter the florescence effects seen upon mannitol binding, suggesting that the cysteine required for activity is not involved in binding. Competition equilibrium dialysis of mannitol with perseitol showed a 50% reduction in mannitol binding at about 40 μM, significantly above the concentration at which fluorescence effects are seen, suggesting that mannitol is sequestered upon binding, but that perseitol is not until higher concentrations are reached. Evidence for mannitol-1-phosphate, but not mannitol or arabitol, binding by NMR was found. Dilution of Enzyme IImtl resulted in a time-dependent decrease in fluorescence emission which approached an equilibrium value. Addition of mannitol showed a smaller increase in fluorescence after equilibrium was reached than immediately following dilution, consistent a with loss of binding sites, The membrane fragment also exhibited this phenomemon. Equilibrium dialysis of concentrated or diluted Enzyme IImtl also showed a loss of binding sites upon dilution, consistent with dissociation of mannitol-binding dimers to non-binding monomers.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

W. Barton Rippon

Second Advisor

Charles Slattery

Third Advisor

Barry L. Taylor

Fourth Advisor

Lawrence Sandberg

Fifth Advisor

Eric Goulbourne

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1988

Date (Title Page)

9-1988

Language

English

Library of Congress/MESH Subject Headings

Escherichia Coli -- analysis; Enzymes -- analysis; Ligands; Mannitol -- physiology

Type

Dissertation

Page Count

viii; 157

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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