Random circular permutation leading to chain disruption within and near α helices in the catalytic chains of aspartate transcarbamoylase:: Effects on assembly, stability, and function

被引:18
作者
Beernink, PT
Yang, YR
Graf, R
King, DS
Shah, SS
Schachman, HK
机构
[1] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
[2] Univ Calif Berkeley, Virus Lab, Berkeley, CA 94720 USA
[3] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA
[4] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
关键词
circular permutation; protein engineering; cooperativity; folding; stability;
D O I
10.1110/ps.39001
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
A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric, As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.
引用
收藏
页码:528 / 537
页数:10
相关论文
共 34 条
[11]   Random circular permutation of DsbA reveals segments that are essential for protein folding and stability [J].
Hennecke, J ;
Sebbel, P ;
Glockshuber, R .
JOURNAL OF MOLECULAR BIOLOGY, 1999, 286 (04) :1197-1215
[12]   EXTENT OF N-TERMINAL METHIONINE EXCISION FROM ESCHERICHIA-COLI PROTEINS IS GOVERNED BY THE SIDE-CHAIN LENGTH OF THE PENULTIMATE AMINO-ACID [J].
HIREL, PH ;
SCHMITTER, JM ;
DESSEN, P ;
FAYAT, G ;
BLANQUET, S .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (21) :8247-8251
[13]   ALLOSTERIC REGULATION OF ASPARTATE TRANSCARBAMOYLASE - CHANGES IN SEDIMENTATION COEFFICIENT PROMOTED BY BISUBSTRATE ANALOG N-(PHOSPHONACETYL)-L-ASPARTATE [J].
HOWLETT, GJ ;
SCHACHMAN, HK .
BIOCHEMISTRY, 1977, 16 (23) :5077-5083
[14]   Influence of primary sequence transpositions on the folding pathways of ribonuclease T1 [J].
Johnson, JL ;
Raushel, FM .
BIOCHEMISTRY, 1996, 35 (31) :10223-10233
[15]   COMPLEX OF N-PHOSPHONACETYL-L-ASPARTATE WITH ASPARTATE CARBAMOYLTRANSFERASE - X-RAY REFINEMENT, ANALYSIS OF CONFORMATIONAL-CHANGES AND CATALYTIC AND ALLOSTERIC MECHANISMS [J].
KE, HM ;
LIPSCOMB, WN ;
CHO, YJ ;
HONZATKO, RB .
JOURNAL OF MOLECULAR BIOLOGY, 1988, 204 (03) :725-747
[16]   MOLSCRIPT - A PROGRAM TO PRODUCE BOTH DETAILED AND SCHEMATIC PLOTS OF PROTEIN STRUCTURES [J].
KRAULIS, PJ .
JOURNAL OF APPLIED CRYSTALLOGRAPHY, 1991, 24 :946-950
[17]  
LIPSCOMB WN, 1994, ADV ENZYMOL RAMB, V68, P67
[18]   Subdomain interactions as a determinant in the folding and stability of T4 lysozyme [J].
Llinás, M ;
Marqusee, S .
PROTEIN SCIENCE, 1998, 7 (01) :96-104
[19]   CORRECT FOLDING OF CIRCULARLY PERMUTED VARIANTS OF A BETA-ALPHA-BARREL ENZYME INVIVO [J].
LUGER, K ;
HOMMEL, U ;
HEROLD, M ;
HOFSTEENGE, J ;
KIRSCHNER, K .
SCIENCE, 1989, 243 (4888) :206-210
[20]   TRANSPOSITION OF PROTEIN SEQUENCES - CIRCULAR PERMUTATION OF RIBONUCLEASE-T1 [J].
MULLINS, LS ;
WESSELING, K ;
KUO, JM ;
GARRETT, JB ;
RAUSHEL, FM .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (13) :5529-5533