Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the nmr spectrum of ribonuclease A in H2O

✍ Scribed by Dinshaw J. Patel; Lita L. Canuel; Clare Woodward; Frank A. Bovey

• Publisher: Wiley (John Wiley & Sons)
• Year: 1975
• Tongue: English
• Weight: 588 KB
• Volume: 14
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1975.360140506

✦ Synopsis

Synopsis

Described herein are proton nmr experiments on chemically modified derivatives of riboniiclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK, of 6.1 in HzO solution. Histidines 48 and 10.5, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate-water solutions. This exchangeable resonance titrates with modified pK,'s and constant area over the above pH range in His-1 19-.1'1-carboxymethylated-RNase A and des-(121-124)-1~Nase A, thus eliminating the imidazole I V ~ proton in the His 119-Asp 121 hydrogen bond. In His-12-~VV1-carboxyniethylated-RNase A, this resonance is also observable, bat broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidaxole ~V I proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 4.5 in the crystal.