The active center is the region where the enzyme reaction occurs, with amino-acid side chains precisely positioned. Using chemical modification and peptide mapping, Moore and Stein pinpointed Histidine-12 and Histidine-119 as essential acid–base catalysts. X-ray crystallography visualized the three-dimensional layout around the active site and led to a mechanism for RNA cleavage. Their work underpins modern enzyme design and inhibitor development.

Moore and Stein digested RNase A with cyanogen bromide and trypsin, separated peptides by ion-exchange chromatography, and determined partial sequences before selectively chemically modifying candidate active-site residues. By identifying peptides whose modification abolished activity and analyzing them by mass spectrometry and spectral methods, they assigned roles to His12, His119, Lys41 and Asp121. Combining pH-dependent kinetics with these data, they proposed a reaction model in which the two histidines act cooperatively as general acid–base catalysts. A 1.9 Å X-ray structure revealed the substrate-binding groove, providing a spatial rationale for the mechanism.

Kinetic analysis of diazo-modification of histidyl residues and pKa shifts indicated a two-step general acid–base mechanism in which His12 deprotonates the 2'-OH and His119 protonates the 5'-O leaving group. The high-resolution X-ray model showed that a His-Lys-Asp cluster forms an electrostatic shielding field that draws in the phosphodiester and stabilizes the transition state. Site-specific ^15N labeling combined with NMR monitored τ- and ε-nitrogen protonation states of the histidines, verifying proton-transfer dynamics during the catalytic cycle.