Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

Abstract

Metallonucleases conduct metal dependent nucleic acid hydrolysis. While metal ions serve in multiple mechanistic capacities in these enzymes, precisely how the attacking water is activated remains unclear for those lacking an obvious general base. All arguments hinge on appropriate pK a 's for active site moieties very close to this species, and measurement of the pK a of a specific water molecule is difficult to access experimentally. Here we describe a computational approach for exploring the local electrostatic influences on the water-derived nucleophile in metallonucleases featuring the common PD…(D/E)xK motif. We utilized UHBD to predict the pK a 's of active site groups, including that of a water molecule positioned to act as a nucleophile. The pK a of a Mg(II)-ligated water molecule hydrogen bonded to the conserved Lys70 in a Mg(II)-PvuII enzyme complex was calculated to be 6.5. The metal and the charge on the Lys group were removed in separate experiments; both resulted in the elevation of the pK a of this water molecule, consistent with contributions from both moieties to lowering this pK a . This behavior is preserved among other PD…(D/E)xK metallonucleases. pK a 's extracted from the pH dependence of the single turnover rate constant are compared to previous experimental data and the above predicted pK a 's.

Catalytic Versatility and Backups in Enzyme Active Sites: The Case of Serum Paraoxonase 1

“…The ligating residues can therefore assist in aligning and activating water for the nucleophilic attack or in protonating the leaving group, as seen in many other metallo-enzymes. [43][44][45] Removal of an individual residue, such as His115, affects catalysis but does not abolish it altogether-primarily because other residues in the catalytic network take over the role of H115. This catalytically rich environment also mediates PON1's catalytic promiscuity.…”

DNA intercalation without flipping in the specific ThaI–DNA complex

“…We note that the question of one versus two or even three metal ions in PD-(D/E)XK restriction endonucleases has never been fully resolved. Like the quest for the precise role of the individual metal ions, it might not have a universally applicable answer ( 2 , 8 , 10 , 55–57 ).…”

“…The two metal ions play distinctly different roles ( 9 ). In the pre-reactive complex, metal ion A positions a solvent molecule for in-line nucleophilic attack and helps to deprotonate it ( 2 , 10 ) ( Figure 1 ). Both metal ions (A and B) coordinate the proS oxygen atom of the phosphate at the site of cleavage, presumably to stabilize the (extra) negative charge in the transition state ( 11 ).…”

“…The aspartate or glutamate in this motif coordinates metal ion A. The lysine is thought to either help to stabilize the extra negative charge in the transition state, or alternatively to act as a base with respect to the attacking water molecule ( 2 , 10 ). The ‘canonical’ arrangement of active site residues in the PD-(D/E)XK nuclease family is not invariable.…”

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

“…Alternately, as occurs in BamHI ( 42 ), Asp74 or Glu75 might act as the general base in the catalytic reaction, assisting in the creation of the hydroxide nucleophile needed for in-line attack on the phosphorus atom, a hypothesized role usually assigned to the lysine (K) of the PD-D/EXK motif ( 43 ). In Vsr, His64 or His69 are positioned to act as the general base rather than lysine; His77 or His78 of AbaSI might act in this way, too.…”