Identification of essential residues in 2 ',3 '-cyclic nucleotide 3 '-phosphodiesterase - Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) catalyzes i n vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides t o produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C- terminal two-thirds of recombinant rat CNP1 identified a region that posses ses the catalytic domain, with further truncations abolishing activity. Pro teolysis and kinetic analysis indicated that this domain forms a compact gl obular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemica l modification studies to identify amino acid residues essential for activi ty. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic a nalysis of cysteine CNP-CF mutants revealed the nonessential role of cystei nes for enzymatic activity. On the other hand, modification studies with di ethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His- 309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K-m values wer e similar. Circular dichroism studies demonstrated that the low catalytic a ctivities of the histidine mutants were not due to gross changes in seconda ry structure. Taken together, these results demonstrate that both histidine s assume critical roles for catalysis.