Cloning, expression and mutagenesis of a subunit contact of rabbit muscle-specific (betabeta) enolase.
The cDNA for rabbit muscle-specific (betabeta) enolase was cloned, sequenced and expressed in Escherichia coli. This betabeta-enolase differs at eight positions from that sequenced by Chin (17). Site-directed mutagenesis was used to change residue 414 from glutamate to leucine, thereby abolishing a salt bridge involved in subunit contacts. Recombinant wild-type and mutant enolase were purified from E. coli and compared to enolase isolated from rabbit muscle. Molecular weights were determined by mass spectrometry. All three betabeta-enolases had similar kinetics, and UV and circular dichroism (CD) spectra. The mutant enolase was far more sensitive to inactivation by pressure, by KCl or EDTA, and by sodium perchlorate. We confirmed, by analytical ultracentrifugation, that the sodium perchlorate inactivation was due to dissociation. DeltaG(o) for dissociation of enolase was decreased from 49.7 kJ/mol for the wild-type enzyme to 42.3 kJ/mol for the mutant. In contrast to the wild-type enzyme, perchlorate inactivation of E414L was accompanied by a small loss of secondary structure.
Kornblatt
Mary Judith
MJ
Enzyme Research Group, Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard W., Montreal, Quebec, Canada H3G 1M8. judithk@vax2.concordia.ca
Zheng
Shu Xian
SX
Lamandé
Noel
N
Lazar
Monique
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase