INVIVO FORMATION OF ACTIVE ASPARTATE TRANSCARBAMOYLASE FROM COMPLEMENTING FRAGMENTS OF THE CATALYTIC POLYPEPTIDE-CHAINS

被引:12
作者
YANG, YR
SCHACHMAN, HK
机构
[1] UNIV CALIF BERKELEY,DEPT MOLEC & CELL BIOL,WENDELL M STANLEY HALL,229 STANLEY HALL,BERKELEY,CA 94720
[2] UNIV CALIF BERKELEY,VIRUS LAB,BERKELEY,CA 94720
关键词
CHAIN OVERLAP; COMPLEMENTATION; FOLDING; OLIGOMER FORMATION FROM FRAGMENTS;
D O I
10.1002/pro.5560020614
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Despite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains. Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed. When the N-terminal peptide (residues 1-242) and the C-terminal peptide (residues 235-310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts. Mixing the two insoluble pellets in 6.5 M urea, followed by a 10-fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8-amino acid redundancy. When the two partial genes were linked into a single transcriptional unit separated by a 15-nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains. The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (V(max)) only slightly less than that of the wild-type C trimer and ATCase. However, K(m) for aspartate exhibited by the C trimer composed of fragmented chains is more than 10-fold larger than that of the wild-type trimer. The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild-type ATCase for which the Hill coefficient is 1.7. K(m) for aspartate as well as K(d) for the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate are significantly higher than the analogous values for wild-type ATCase. Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild-type ATCase.
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收藏
页码:1013 / 1023
页数:11
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