| Autor: |
De Vos, Stefan, Doumen, Jan, Langhorst, Ulrike, Steyaert, Jan |
| Zdroj: |
JMB Online (Journal of Molecular Biology); January 1998, Vol. 275 Issue: 4 p651-661, 11p |
| Abstrakt: |
His92 of Ribonuclease T1combines functional and structural features involving both imidazole nitrogens. To evaluate the use of Asn and Gln substitutions in dissecting the properties of histidines, we analysed the consequences of the His92Gln and His92Asn substitutions on the enzyme’s structure, function, and conformational stability by protein engineering and X-ray crystallographic methods. In the X-ray structures of wild-type and His92Gln RNase T1in complex with 2′-GMP the His92-Nϵ 2and Gln92-Nϵ2atoms are isosterically equivalent. Similarly, the His92Nδ1H…OAsn99 hydrogen bond observed in wild-type is replaced by an equivalent Asn92Nδ 2H…OAsn99 in the His92Asn mutant structure. Double mutant cycles at a single position were used to analyse the intermolecular and intramolecular interactions of the exchangeable proton and the individual histidine nitrogens. Urea denaturation measurements as a function of pH revealed that the exchangeable proton of His92, rather than its imidazole ring is contributing about 1 kcal/mol to the conformational stability of RNase T1. The stabilizing and the destabilizing effects of the (His → Gln) and the (His → Asn) mutations on urea denaturation of RNase T1at pH 9.0 suggest that the unprotonated Nδ1and Nϵ2atoms contribute in a compensating way to the conformational stability of RNase T1. A comparative study of the kinetics of all mutants suggests that the protonated His92 imidazole is a strictly co-operative catalytic device. |
| Databáze: |
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