| Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies. | |
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MedLine Citation:
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PMID: 8756679 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. All of the variants still retained significant activity, and all could be purified to homogeneity. FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme. There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant. This frequency is appropriate to a cysteine S-H stretching mode. It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized. Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain). The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme. The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 [Baburina et al. (1994) Biochemistry 33, 5630-5635] is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase. This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid. |
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Authors:
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I Baburina; D J Moore; A Volkov; A Kahyaoglu; F Jordan; R Mendelsohn |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Biochemistry Volume: 35 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 1996 Aug |
Date Detail:
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Created Date: 1996-09-30 Completed Date: 1996-09-30 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 10249-55 Citation Subset: IM |
Affiliation:
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Department of Chemistry, Rutgers, State University of New Jersey, Newark 07102, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Cysteine
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chemistry* DNA Primers Enzyme Activation Hydrogen-Ion Concentration Isoelectric Focusing Mutagenesis, Site-Directed Pyruvate Decarboxylase / chemistry*, genetics, metabolism Saccharomyces cerevisiae / enzymology* Spectroscopy, Fourier Transform Infrared Substrate Specificity |
| Grant Support | |
ID/Acronym/Agency:
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GM29864/GM/NIGMS NIH HHS; GM50380/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/DNA Primers; 52-90-4/Cysteine; EC 4.1.1.1/Pyruvate Decarboxylase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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