Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis

被引:13
作者
Pasquo, A
Britton, KL
Stillman, TJ
Rice, DW
Colfen, H
Harding, SE
Scandurra, R
Engel, PC
机构
[1] UNIV ROMA LA SAPIENZA,DIPARTIMENTO SCI BIOCHIM A ROSSI FANELLI,I-00185 ROME,ITALY
[2] UNIV SHEFFIELD,KREBS INST,DEPT MOL BIOL & BIOTECHNOL,SHEFFIELD S10 2UH,S YORKSHIRE,ENGLAND
[3] UNIV NOTTINGHAM,DEPT APPL BIOCHEM & FOOD SCI,SUTTON LE12 5RD,SURREY,ENGLAND
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 1996年 / 1297卷 / 02期
关键词
glutamate dehydrogenase; subunit interaction; site-directed mutagenesis; (C-symbiosum);
D O I
10.1016/S0167-4838(96)00017-9
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
By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue, Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots, The antibody was used to monitor purification of the inactive protein, F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87 000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD(+) completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients s(20,w) of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280 000 +/- 5000 g/mol, consistent with the hexameric structure for the wild-type protein and 135 000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98 000) and a trimer (147 000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 1/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 1/g, Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species, Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.
引用
收藏
页码:149 / 158
页数:10
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