Common Patterns of Hydrolysis Initiation in P-loop Fold Nucleoside Triphosphatases

The P-loop fold nucleoside triphosphate (NTP) hydrolases (also known as Walker NTPases) function as ATPases, GTPases, and ATP synthases, are often of medical importance, and represent one of the largest and evolutionarily oldest families of enzymes. There is still no consensus on their catalytic mechanism. To clarify this, we performed the first comparative structural analysis of more than 3100 structures of P-loop NTPases that contain bound substrate Mg-NTPs or their analogues. We proceeded on the assumption that structural features common to these P-loop NTPases may be essential for catalysis. Our results are presented in two articles. Here, in the first, we consider the structural elements that stimulate hydrolysis. Upon interaction of P-loop NTPases with their cognate activating partners (RNA/DNA/protein domains), specific stimulatory moieties, usually Arg or Lys residues, are inserted into the catalytic site and initiate the cleavage of gamma phosphate. By analyzing a plethora of structures, we found that the only shared feature was the mechanistic interaction of stimulators with the oxygen atoms of gamma-phosphate group, capable of causing its rotation. One of the oxygen atoms of gamma phosphate coordinates the cofactor Mg ion. The rotation must pull this oxygen atom away from the Mg ion. This rearrangement should affect the properties of the other Mg ligands and may initiate hydrolysis according to the mechanism elaborated in the second article

Common Mechanism of Activated Catalysis in P-loop Fold Nucleoside Triphosphatases—United in Diversity

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 (AspWB) 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 AspWB. 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 AspWB 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

Hypoxia affects neprilysin expression through caspase activation and an APP intracellular domain-dependent mechanism

While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer’s disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8 and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischemia leading to a deficit of Aβ clearance and increasing the risk of development of AD