Abstract
To clarify the obscure hydrolysis mechanism of ubiquitous P-loop-fold nucleoside triphosphatases (Walker NTPases), we analysed the structures of 3136 catalytic sites with bound Mg-NTP complexes or their analogues. Our results are presented in two articles; here, in the second of them, we elucidated whether the Walker A and Walker B sequence motifs-common to all P-loop NTPases-could be directly involved in catalysis. We found that the hydrogen bonds (H-bonds) between the strictly conserved, Mg-coordinating Ser/Thr of the Walker A motif ([Ser/Thr](WA)) and aspartate of the Walker B motif (Asp(WB)) are particularly short (even as short as 2.4 ångströms) in the structures with bound transition state (TS) analogues. Given that a short H-bond implies parity in the pKa values of the H-bond partners, we suggest that, in response to the interactions of a P-loop NTPase with its cognate activating partner, a proton relocates from [Ser/Thr](WA) to Asp(WB). The resulting anionic [Ser/Thr](WA) alkoxide withdraws a proton from the catalytic water molecule, and the nascent hydroxyl attacks the gamma phosphate of NTP. When the gamma-phosphate breaks away, the trapped proton at Asp(WB) passes by the Grotthuss relay via [Ser/Thr](WA) to beta-phosphate and compensates for its developing negative charge that is thought to be responsible for the activation barrier of hydrolysis.