| | |  | | | | Record from Web of Science® | | | | | | |
| SACCHAROMYCES-CEREVISIAE PHOSPHOENOLPYRUVATE CARBOXYKINASE - REVISED AMINO-ACID-SEQUENCE, SITE-DIRECTED MUTAGENESIS, AND MICROENVIRONMENT CHARACTERISTICS OF CYSTEINE-365 AND CYSTEINE-458 |
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| Author(s): KRAUTWURST H, ENCINAS MV, MARCUS F, LATSHAW SP, KEMP RG, FREY PA, CARDEMIL E |
| Source: BIOCHEMISTRY Volume: 34 Issue: 19 Pages: 6382-6388 Published: MAY 16 1995 |
| Times Cited: 27 References: 44 |
| Abstract: Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys(365) Or Cys(458); however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence, Several of these descrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys(364) and Cys(457) correspond to Cys(365) and Cys(458). The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys(458) is 7-fold more reactive toward the sulfhydryl-directed probe than Cys(365), while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV SANTIAGO CHILE, FAC QUIM & BIOL, FAC CIENCIAS QUIM, SANTIAGO, CHILE
2. CHIRON CORP, EMERYVILLE, CA 94608 USA
3. UNIV HLTH SCI CHICAGO MED SCH, DEPT BIOL CHEM, N CHICAGO, IL 60064 USA
4. UNIV WISCONSIN, INST ENZYME RES, MADISON, WI 53705 USA |
| Publisher: AMER CHEMICAL SOC, PO BOX 57136, WASHINGTON, DC 20037-0136 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: QZ716 |
| ISSN: 0006-2960 |
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