Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI

Homing endonucleases are distinguished by their ability to catalyze the cle avage of double-stranded DNA with extremely high specificity. I-PpoI endonu clease, a homing endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) of known three-dimensional structure that cata lyzes the cleavage of a long target DNA sequence (15 base pairs). Here, a d etailed chemical mechanism for catalysis of DNA cleavage by I-PpoI endonucl ease is proposed and tested by creating six variants in which active-site r esidues are replaced with alanine. The side chains of three residues (Arg61 , His98, and Asn119) are found to be important for efficient catalysis of D NA cleavage. This finding is consistent with the proposed mechanism in whic h His98 abstracts a proton from an attacking water molecule bound by an adj acent phosphoryl oxygen, Arg61 and Asn119 stabilize the pentavalent transit ion state, and Asn119 also binds to the essential divalent metal cation (e. g., Mg2+ ion), which interacts with the S'-oxygen leaving group. Because Mg 2+ is required for cleavage of a substrate with a good leaving group (p-nit rophenolate), Mg2+ likely stabilizes the pentavalent transition state. The DH-dependence of k(cat) for catalysis by I-PpoI reveals a macroscopic pK(a) of 8.4 for titratable groups that modulate product release. I-PpoI appears to be unique among known restriction endonucleases and homing endonuclease s in its use of a histidine residue to activate the attacking water molecul e for in-line displacement of the 3'-leaving group.