Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI.
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.
Mannino
S J
SJ
Department of Biochemistry, Department of Chemistry, University of Wisconsin-Madison 53706, USA.
Jenkins
C L
CL
Raines
R T
RT
D002384:Catalysis; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D009841:Oligonucleotides; C117362:deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate); D004247:DNA; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; D013936:Thymidine