Investigations of the kinetic mechanism of kanamycin nucleotidyltransferase and the binding pockets of phosphotriesterase /
The kinetic and chemical mechanism of kanamycin nucleotidyltransferase (KNTase) has been determined to be an ordered Bi-Bi kinetic mechanism, with the aminoglycoside antibiotic being the first substrate to bind to the enzyme and the adenylylated antibiotic being the last product to be released from...
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=728331691&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The kinetic and chemical mechanism of kanamycin nucleotidyltransferase (KNTase) has been determined to be an ordered Bi-Bi kinetic mechanism, with the aminoglycoside antibiotic being the first substrate to bind to the enzyme and the adenylylated antibiotic being the last product to be released from the active site of KNTase. The overall rate of the enzymatic reaction appears to be limited by the release of the product. Site-directed mutagenesis studies have shown that Glu 145 is essential for antibiotic binding as well as catalysis. Studies with chiral organophosphotriesters show that the wild type phosphotriesterase (PTE) prefers Sp enantiomer over Rp enantiomer by 10 to 90-fold. The crystal structure of PTE reveals three binding pockets (small, large, and leaving group subsites) within the active site of the enzyme. These studies have shown that reduction of the small subsite with G60A significantly increases the preference for the faster Sp enantiomer. Enlargement of the small subsite with glycine and alanine mutants of Ile 106, Phe 132, and Ser 308 resulted in a reduction or elimination of the stereoselectivity observed with the wild type enzyme toward chiral substrates. Enlargement of the small subsite and reduction of the large subsite simultaneously with multiple-site mutations at Ile 106, Phe 132, Ser 308 and His 257 resulted in up to 460-fold preference for the Rp enantiomer. The wild type enzyme exhibits a 20-fold preference for the Rp enantiomer of isopropyl p-nitrophenyl methylphosphonate. The chiral preference for the Rp enantiomer of the substrate was significantly enhanced by reducing the small subsite with G60A or G60S. The stereoselectivity can be eliminated by enlarging the small subsite with I106A and I106A/F132A, or by reducing the large subsite with H254F, H254Y, H257F, H257W, L271F, L271Y, L271W, and M317Y. The stereoselectivity can be reversed by up to 370-fold by altering the small and large subsites simultaneously with I106A/H257Y, I106A/F132A/H257Y, and I106A/F132A/H257W. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiv, 203 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 195-201). |