Abstract：
:Homing endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with extremely high specificity. I-PpoI endonuclease, a homing endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) of known three-dimensional structure that catalyzes the cleavage of a long target DNA sequence (15 base pairs). Here, a detailed chemical mechanism for catalysis of DNA cleavage by I-PpoI endonuclease is proposed and tested by creating six variants in which active-site residues are replaced with alanine. The side chains of three residues (Arg61, His98, and Asn119) are found to be important for efficient catalysis of DNA cleavage. This finding is consistent with the proposed mechanism in which His98 abstracts a proton from an attacking water molecule bound by an adjacent phosphoryl oxygen, Arg61 and Asn119 stabilize the pentavalent transition state, and Asn119 also binds to the essential divalent metal cation (e.g., Mg(2+) ion), which interacts with the 3'-oxygen leaving group. Because Mg(2+) is required for cleavage of a substrate with a good leaving group (p-nitrophenolate), Mg(2+) likely stabilizes the pentavalent transition state. The pH-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 endonucleases in its use of a histidine residue to activate the attacking water molecule for in-line displacement of the 3'-leaving group.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mannino SJ,Jenkins CL,Raines RTdoi
10.1021/bi991452vsubject
Has Abstractpub_date
1999-12-07 00:00:00pages
16178-86issue
49eissn
0006-2960issn
1520-4995pii
bi991452vjournal_volume
38pub_type
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