ROLE OF A CARBOXYL-TERMINAL HELIX IN THE ASSEMBLY, INTERCHAIN INTERACTIONS, AND STABILITY OF ASPARTATE TRANSCARBAMOYLASE

被引:15
作者
PETERSON, CB
SCHACHMAN, HK
机构
[1] UNIV CALIF BERKELEY,DEPT MOLEC & CELL BIOL,WENDELL M STANLEY HALL,BERKELEY,CA 94720
[2] UNIV CALIF BERKELEY,VIRUS LAB,BERKELEY,CA 94720
关键词
TRUNCATED POLYPEPTIDE CHAINS; SITE-DIRECTED MUTAGENESIS; DIFFERENTIAL SCANNING CALORIMETRY;
D O I
10.1073/pnas.88.2.458
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The six individual catalytic polypeptide chains within the two catalytic trimers of Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) are folded into two discrete structural domains interconnected in part by helix 12, which comprises residues 285-305 and is located near the carboxyl terminus of the chain. The essential role of this helix in folding of the chains and their assembly into ATCase was demonstrated by introducing a stop codon at the position corresponding to amino acid 284, 291, or 299. Cells containing these mutations are pyrimidine auxotrophs lacking ATCase-like protein in cell extracts. In contrast, stable active enzyme is formed from chains truncated at position 306 or 307, showing that all 310 amino acids are not required for assembly. Replacements of Gln-288, Asn-291, Arg-296, and Ala-298 were introduced to assess the effect of alterations within helix 12 on protein stability. Stability of the trimers was measured both by differential scanning microcalorimetry and by the rate of exchange of chains at 4-degrees-C when mutant trimers were incubated with succinylated wild-type trimers. Melting temperatures of the mutant trimers spanned a range of more than 20-degrees-C, with a few higher and others lower than that of wild-type trimers. Large changes in interchain interaction energies were observed for the trimers, but there was no direct correlation between the ease of dissociation of the trimers and their thermal stability. Calorimetry on the mutant holoenzymes revealed alterations in the interactions between trimers and regulatory subunits within the intact enzymes. The striking changes in stability of both trimers and holoenzymes demonstrated that effects of relatively localized amino acid replacements in helix 12 are manifested by indirect global alterations propagated throughout the structure.
引用
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页码:458 / 462
页数:5
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