Reassignment of the active site histidines in ribonuclease A by selective deuteration studies

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

• Publisher: Wiley (John Wiley & Sons)
• Year: 1975
• Tongue: English
• Weight: 493 KB
• Volume: 14
• Category: Article
• ISSN: 0006-3525
• DOI: 10.1002/bip.1975.360140508

✦ Synopsis

Abstract

The C~2~H resonance of the active site histidine residue designated AS‐2, which has the lower p__K__~a~ of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP‐3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS‐2 is absent in the spectrum of RNase S′ reconstituted using S‐peptide deuterated at the C~2~ of His 12, and in that of the RNase S′‐CMP‐3′ complex. We thus demonstrate that histidine AS‐2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C~2~H resonances made earlier by other authors.