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Enzymatic hydrolysis of the amide bond : mutagenic studies of the mechanisms of [alpha]-lytic protease and [beta]-lactamase

Citation

Emerling, Michael Roy (1991) Enzymatic hydrolysis of the amide bond : mutagenic studies of the mechanisms of [alpha]-lytic protease and [beta]-lactamase. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06202007-140449

Abstract

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The enzymatic hydrolysis of amide bonds was studied in two systems by site-specific mutagenic techniques. In the first study, I developed an expression system for the serine protease, [alpha]-lytic protease, from Lysobacter enzymogenes 496, using a previously constructed synthetic gene, which contained numerous unique restriction sites. Since the wild type enzyme is expressed as a zymogen, which is self-processed into the mature proteolytic enzyme, a unique chimeric expression system utilizing the concomitant expression of the pro-domain from the wild type enzyme was created. The system expresses the properly-folded, mature protease-domain of the enzyme, thus allowing for production of active-site mutants of [alpha]-lytic protease, that otherwise could not be obtained in the mature form.

The ability of the enzymatic machinery to enhance the nucleophilicity of a chemical group other than the active-site serine hydroxyl was investigated through the mutation of serine 195 to an alanine. The removal of the serine hydroxyl was hypothesized to provide sufficient volume in the active site to allow a water molecule to bind and possibly function as the attacking nucleophile in the hydrolysis. The structure of the enzyme would be minimally disturbed by the removal of the single atom. The mutant enzyme was assayed for activity on the serine protease inhibitor diethyl [...]-nitrophenyl phosphate. No enzymatic hydrolysis of the substrate was detected. Analysis of structural constraints of the enzyme suggests that a serine 195 glycine mutation might provide a more hydrophilic environment in the active site for the binding of a water molecule.

In the second study, I investigated the mechanism of RTEM-1 [beta]-lactamase, an enzyme which hydrolyzes the amide bond of [beta]-lactam antibiotics, conferring antibiotic resistance to bacterial cells. Serine 130, a conserved active-site residue in the class A [beta]-lactamases, has been proposed to be involved in positioning the conserved lysine 234 through a hydrogen bond interaction (Moews et al. (1990) Proteins 7 156). The function of lysine 234 is known to be one solely of substrate binding (D. M. Long (1991) Ph.D. Thesis, California Institute of Technology). I performed site-saturation mutagenesis of serine 130, and the resulting 20 mutant enzymes were assayed for the ability to confer resistance to E. coli towards several [beta]-lactam antibiotics. Four mutants (Ser 130 Gly, Ser 130 Thr, Ser 130 Asn, and Ser 130 Gln) conferred notable resistance to the penam antibiotics. These four mutants were purified to homogeneity, and the steady-state kinetic parameters for hydrolysis of benzylpenicillin were measured for each. The values of KM for all four mutants were no more than ten-fold more than the wild type value. However, values of kcat for the four mutants were decreased at least 1000-fold from that of the wild type, demonstrating clearly the involvement of serine 130 in catalysis. These results, along with an analogy to structural mutants of Thr 157 in T4 lysozyme, suggest how the four serine 130 mutants might maintain the native hydrogen bonding interactions of the serine with lysine 234, as well as participate in the catalytic mechanism.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Richards, John H.
Thesis Committee:
  • Unknown, Unknown
Defense Date:29 April 1991
Record Number:CaltechETD:etd-06202007-140449
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06202007-140449
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2663
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:16 Jul 2007
Last Modified:26 Dec 2012 02:53

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