3BIR

DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 BY ASN AND GLN SUBSTITUTIONS

Summary for 3BIR

• Entry DOI: 10.2210/pdb3bir/pdb
• Descriptor: RIBONUCLEASE T1, CALCIUM ION, GUANOSINE-2'-MONOPHOSPHATE, ... (4 entities in total)
• Functional Keywords: endonuclease, hydrolase, ribonuclease t1, mutation
• Biological source: Aspergillus oryzae
• Total number of polymer chains: 1
• Total formula weight: 11514.03

Authors

Doumen, J.,Steyaert, J. (deposition date: 1997-06-27, release date: 1997-12-31, Last modification date: 2024-11-06)

Primary citation

De Vos, S.,Doumen, J.,Langhorst, U.,Steyaert, J.
Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position.
J.Mol.Biol., 275:651-661, 1998

PubMed Abstract: His92 of Ribonuclease T1 combines 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 T1 in complex with 2'-GMP the His92-N epsilon 2 and Gln92-N epsilon 2 atoms are isosterically equivalent. Similarly, the His92N delta 1H...OAsn99 hydrogen bond observed in wild-type is replaced by an equivalent Asn92N delta 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 T1 at pH 9.0 suggest that the unprotonated N delta 1 and N epsilon 2 atoms 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.

PubMed: 9466938
DOI: 10.1006/jmbi.1997.1480

Experimental method

X-RAY DIFFRACTION (1.8 Å)