Studies on the binuclear metal center of phosphotriesterase and hydrolysis of phosphate esters /
The mechanism of the assembly of the binuclear metal center of phosphotriesterase from Pseudomonas diminuta was investigated. The activation kinetics were shown to be pseudo first-order, with the observed rate constant proportional to the concentration of carbon dioxide and inversely proportional t...
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| Format: | Thesis Book |
| Language: | English |
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[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=727726431&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The mechanism of the assembly of the binuclear metal center of phosphotriesterase from Pseudomonas diminuta was investigated. The activation kinetics were shown to be pseudo first-order, with the observed rate constant proportional to the concentration of carbon dioxide and inversely proportional to the concentration of the metal ion. These patterns are consistent with an ordered mechanism of assembly in which carbon dioxide reacts with the apoenzyme, and the two metal ions bind subsequently to this complex. The metal dissociation constants and the dissociation rate constants were measured. The data showed that metal binding to the enzyme is very tight (K[D]₁K[D]₂ = 3.6 x 10⁻²¹ M² and k₀[ff] = 1.5 þ 10⁻³ min⁻¹ for Zn(II)). A comparison study using the K169A mutant enzyme showed that the carbamate bridge is required for both efficient catalysis and stability of the enzyme-metal complex. It was shown that phosphotriesterase is capable of catalyzing the hydrolysis of phosphodiesters with a rate enhancement of >10⁸-fold. Upon the addition of various alkyl amines to the reaction mixture, the k[cat]/K[m] for the phosphodiester ethyl 4-nitrophenylphosphate increased up to 200-fold. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement. The primary and secondary ¹⁸O isotope effects of O, O-diethyl phosphorylcholine were measured in an effort to probe the transition state structure of the chemical hydrolysis of phosphotriester. The results suggest that the reaction mechanism for the chemical hydrolysis of phosphotriester is associative without formation of a distinctive pentacoordinate intermediate, and that the reaction occurs in a concerted but asynchronous fashion. The large primary oxygen isotope effect for the hydrolysis of this phosphotriester substrate with a poor leaving group indicates a late, product-like transition state. Mutants of the second sphere residues of PTE were constructed and characterized. The mutants showed significantly compromised catalytic activity and metal binding. A comparison study of the second sphere residues of PTE and related proteins showed little similarity, suggesting a distal evolutionary ancestry and different catalytic functions. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xii, 129 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 120-126). |