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| Domain behavior during the folding of a thermostable phosphoglycerate kinase |
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| Author(s): Parker MJ, Spencer J, Jackson GS, Burston SG, Hosszu LLP, Craven CJ, Waltho JP, Clarke AR |
| Source: BIOCHEMISTRY Volume: 35 Issue: 49 Pages: 15740-15752 Published: DEC 10 1996 |
| Times Cited: 28 References: 40 |
| Abstract: Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monomeric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain ex-helix (residues 170-185). The molecule folds to the native state in three stages. In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol(-1) and the C-domain by 3.3 kcal mol(-1) over their respective unfolded conformations. The N-domain then converts to a folded state at a rate of 1.2 s(-1) (Delta G(I-F) = 3.8 kcal mol(-1)), followed by the C-domain at 0.032 s(-1) (Delta G(I-F) = 12.1 kcal mol(-1)). It is this last step that limits the rate of acquisition of enzyme activity. In the dynamics of unfolding in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s(-1), some 10(7) times faster than the C-domain. Consequently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a native-like C-domain. In a conventional sense, therefore, the folding/unfolding kinetics of bsPGK can be described as random order. Consistent with these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394. A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relatively weak, contributing similar to 1.4 kcal mol-l to the stability of the active enzyme. The only interactions which folding pathways are those within domain cores. Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but do not constitute a separate step in the folding mechanism Intriguingly, the most pronounced effect of interdomain contacts on the kinetics of folding is inhibitory; the presence of the C-domain appearing to reduce the effective rate of acquisition of native structure within the N-domain. |
| Document Type: Article |
| Language: English |
| Reprint Address: Parker, MJ (reprint author), UNIV BRISTOL, SCH MED SCI, DEPT BIOCHEM, BRISTOL BS8 1TD, AVON ENGLAND |
Addresses:
1. UNIV SHEFFIELD, DEPT MOL BIOL & BIOTECHNOL, KREBS INST BIOMOLEC RES, SHEFFIELD S10 2UH, S YORKSHIRE ENGLAND |
| Publisher: AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: VX510 |
| ISSN: 0006-2960 |
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