Understanding the role of P-type ATPases in regulating pollen fertility and development in pigeonpea.

The P-type ATPase superfamily genes are the cation and phospholipid pumps that transport ions across the membranes by hydrolyzing ATP. They are involved in a diverse range of functions, including fundamental cellular events that occur during the growth of plants, especially in the reproductive organs. The present work has been undertaken to understand and characterize the P-type ATPases in the pigeonpea genome and their potential role in anther development and pollen fertility. A total of 59 P-type ATPases were predicted in the pigeonpea genome. The phylogenetic analysis classified the ATPases into five subfamilies: eleven P1B, eighteen P2A/B, fourteen P3A, fifteen P4, and one P5. Twenty-three pairs of P-type ATPases were tandemly duplicated, resulting in their expansion in the pigeonpea genome during evolution. The orthologs of the reported anther development-related genes were searched in the pigeonpea genome, and the expression profiling studies of specific genes via qRT-PCR in the pre- and post-meiotic anther stages of AKCMS11A (male sterile), AKCMS11B (maintainer) and AKPR303 (fertility restorer) lines of pigeonpea was done. Compared to the restorer and maintainer lines, the down-regulation of CcP-typeATPase22 in the post-meiotic anthers of the male sterile line might have played a role in pollen sterility. Furthermore, the strong expression of CcP-typeATPase2 in the post-meiotic anthers of restorer line and CcP-typeATPase46, CcP-typeATPase51, and CcP-typeATPase52 in the maintainer lines, respectively, compared to the male sterile line, clearly indicates their potential role in developing male reproductive organs in pigeonpea.

P-type calcium ATPases play important roles in biotic and abiotic stress signaling.

MAIN CONCLUSION: Knowledge of Ca2+-ATPases is imperative for improving crop quality/ food security, highly threatened due to global warming. Ca2+-ATPases modulates calcium, essential for stress signaling and modulating growth, development, and immune activities. Calcium is considered a versatile secondary messenger and essential for short- and long-term responses to biotic and abiotic stresses in plants. Coordinated transport activities from both calcium influx and efflux channels are required to generate cellular calcium signals. Various extracellular stimuli cause an induction in cytosolic calcium levels. To cope with such stresses, it is important to maintain intracellular Ca2+ levels. Plants need to evolve efficient efflux mechanisms to maintain Ca2+ ion homeostasis. Plant Ca2+-ATPases are members of the P-type ATPase superfamily and localized in the plasma membrane and endoplasmic reticulum (ER). They are required for various cellular processes, including plant growth, development, calcium signaling, and even retorts to environmental stress. These ATPases play an essential role in Ca2+ homeostasis and are actively involved in Ca2+ transport. Plant Ca2+-ATPases are categorized into two major classes: type IIA and type IIB. Although these two classes of ATPases share similarities in protein sequence, they differ in their structure, cellular localization, and sensitivity to inhibitors. Due to the emerging role of Ca2+-ATPase in abiotic and biotic plant stress, members of this family may help promote agricultural improvement under stress conditions. This review provides a comprehensive overview of P-type Ca2+-ATPase, and their role in Ca2+ transport, stress signaling, and cellular homeostasis focusing on their classification, evolution, ion specificities, and catalytic mechanisms. It also describes the main aspects of the role of Ca2+-ATPase in transducing signals during plant biotic and abiotic stress responses and its role in plant development and physiology.

The A subunit of vacuolar H+-ATPase gene (CmVHA-A) plays opposite roles in plant growth and drought tolerance of chrysanthemum under different growing conditions.

As the most prominent proton pumps in plants, vacuolar H+-ATPases (VHAs) comprise multiple subunits that are important for physiological processes and stress tolerance in plants. However, few studies on the roles of subunit genes of VHAs in chrysanthemum have been reported to date. In this study, the gene of A subunit of V-ATPase in chrysanthemum (CmVHA-A) was cloned and identified. CmVHA-A was conserved with VHA-A proteins from other plants. Expression analysis showed that CmVHA-A was highly expressed in most tissues of chrysanthemum except for the flower bud, and was readily induced by polyethylene glycol (PEG) treatment. Functional analysis demonstrated that CmVHA-A exerted a negative influence on the growth and development of shoot and root of chrysanthemum under normal conditions. RNA-sequencing (RNA-seq) analysis revealed the possible explanations for phenotypic differences between transgenic and wild-type (WT) plants. Under drought conditions, CmVHA-A positively affected the drought tolerance of chrysanthemum by enhancing antioxidase activity and alleviating photosynthetic disruption. Overall, CmVHA-A plays opposite roles in plant growth and drought tolerance of chrysanthemums under different growing conditions.

Identification of a Catalytic Lysine Residue Conserved Among GHKL ATPases: MutL, GyrB, and MORC.

DNA mismatch repair endonuclease MutL is a member of GHKL ATPase superfamily. Mutations of MutL homologs are causative of a hereditary cancer, Lynch syndrome. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although involvement of a basic residue had not been conceived in the catalytic mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is catalyzed by a residue with an alkaline pKa. Analyses of mutant aqMutLs showed that Lys79 is the catalytic residue, and the corresponding residues were confirmed to be critical for activities of human MutL homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations. Furthermore, it was confirmed that the catalytic lysine residue is conserved among DNA gyrases and microrchidia ATPases, other members of GHKL ATPases, indicating that the catalytic mechanism proposed here is applicable to these members of the superfamily.

Understanding the dynamic design of the spliceosome.

The spliceosome catalyzes the splicing of pre-mRNAs. Although the spliceosome evolved from a prokaryotic self-splicing intron and an associated protein, it is a vastly more complex and dynamic ribonucleoprotein (RNP) whose function requires at least eight ATPases and multiple RNA rearrangements. These features afford stepwise opportunities for multiple inspections of the intron substrate, coupled with spliceosome disassembly for substrates that fail inspection. Early work using splicing-defective pre-mRNAs or small nuclear (sn)RNAs in Saccharomyces cerevisiae demonstrated that such checks could occur in catalytically active spliceosomes. We review recent results on pre-mRNA splicing in various systems, including humans, suggesting that earlier steps in spliceosome assembly are also subject to such quality control. The inspection-rejection framework helps explain the dynamic nature of the spliceosome.

Involvement of nucleus accumbens SERCA2b in methamphetamine-induced conditioned place preference.

Methamphetamine (METH) is a highly addictive psycho-stimulant that induces addictive behaviour by stimulating increased dopamine release in the nucleus accumbens (NAc). The sarco/endoplasmic reticulum calcium ion transport ATPases (SERCA or ATP2A) is a calcium ion (Ca2+) pump in the endoplasmic reticulum (ER) membrane. SERCA2b is a SERCA subtype mainly distributed in the central nervous system. This study used conditioned place preference (CPP), a translational drug reward model, to observe the effects of SERCA and SERCA2b on METH-CPP in mice. Result suggested that the activity of SERCA was significantly decreased in NAc after METH-CPP. Intraperitoneal SERCA agonist CDN1163 injection or bilateral CDN1163 microinjection in the NAc inhibited METH-CPP formation. SERCA2b overexpression by the Adeno-associated virus can reduce the DA release of NAc and inhibit METH-CPP formation. Although microinjection of SERCA inhibitor thapsigargin in the bilateral NAc did not significantly aggravate METH-CPP, interference with SERCA2b expression in NAc by adeno-associated virus increased DA release and promoted METH-CPP formation. METH reduced the SERCA ability to transport Ca2+ into the ER in SHSY5Y cells in vitro, which was reversed by CDN1163. This study revealed that METH dysregulates intracellular calcium balance by downregulating SERCA2b function, increasing DA release in NAc and inducing METH-CPP formation. Drugs that target SERCA2b may have the potential to treat METH addiction.

Proton pump inhibitors enhance macropinocytosis-mediated extracellular vesicle endocytosis by inducing membrane v-ATPase assembly.

Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.

Functional and in silico analysis of ATP8A2 and other P4-ATPase variants associated with human genetic diseases.

P4-ATPases flip lipids from the exoplasmic to cytoplasmic leaflet of cell membranes, a property crucial for many biological processes. Mutations in P4-ATPases are associated with severe inherited and complex human disorders. We determined the expression, localization and ATPase activity of four variants of ATP8A2, the P4-ATPase associated with the neurodevelopmental disorder known as cerebellar ataxia, impaired intellectual development and disequilibrium syndrome 4 (CAMRQ4). Two variants, G447R and A772P, harboring mutations in catalytic domains, expressed at low levels and mislocalized in cells. In contrast, the E459Q variant in a flexible loop displayed wild-type expression levels, Golgi-endosome localization and ATPase activity. The R1147W variant expressed at 50% of wild-type levels but showed normal localization and activity. These results indicate that the G447R and A772P mutations cause CAMRQ4 through protein misfolding. The E459Q mutation is unlikely to be causative, whereas the R1147W may display a milder disease phenotype. Using various programs that predict protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments, suggesting that such analysis is useful in identifying protein misfolding disease-associated variants.

The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa.

Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1-a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.

DPCD is a regulator of R2TP in ciliogenesis initiation through Akt signaling.

R2TP is a chaperone complex consisting of the AAA+ ATPases RUVBL1 and RUVBL2, as well as RPAP3 and PIH1D1 proteins. R2TP is responsible for the assembly of macromolecular complexes mainly acting through different adaptors. Using proximity-labeling mass spectrometry, we identified deleted in primary ciliary dyskinesia (DPCD) as an adaptor of R2TP. Here, we demonstrate that R2TP-DPCD influences ciliogenesis initiation through a unique mechanism by interaction with Akt kinase to regulate its phosphorylation levels rather than its stability. We further show that DPCD is a heart-shaped monomeric protein with two domains. A highly conserved region in the cysteine- and histidine-rich domains-containing proteins and SGT1 (CS) domain of DPCD interacts with the RUVBL2 DII domain with high affinity to form a stable R2TP-DPCD complex both in cellulo and in vitro. Considering that DPCD is one among several CS-domain-containing proteins found to associate with RUVBL1/2, we propose that RUVBL1/2 are CS-domain-binding proteins that regulate complex assembly and downstream signaling.

Childhood-related neural genotype-phenotype in ATP1A3 mutations: comprehensive analysis.

BACKGROUND: ATP1A3 is a gene that encodes the ATPase Na + /K + transporting subunit alpha-3 isoenzyme that is widely expressed in GABAergic neurons. It maintains metabolic balance and neurotransmitter movement. These pathways are essential for the proper functioning of the nervous system. A mutation in the ATP1A3 gene demonstrates remarkable genotype-phenotype heterogeneity. OBJECTIVES: To provide insight into patients with ATP1A3 mutation. MATERIAL AND METHODS: These cases were identified using next generation sequencing. The patients' clinical and genetic data were retrieved. Detailed revision of the literature was conducted to illustrate and compare findings. The clinical, genetical, neuroimaging, and electrophysiological data of all pediatric patients were extracted. RESULTS: The study included 14 females and 12 males in addition to two novel females cases. Their mean current age is 6.3 ± 4.24 years. There were 11.54% preterm pregnancies with 5 cases reporting pregnancy complications. Mean age of seizure onset was 1.07 ± 1.06 years. Seizure semiology included generalized tonic-clonic, staring spells, tonic-clonic, and others. Levetiracetam was the most frequently used Anti-seizure medication. The three most frequently reported classical symptoms included alternating hemiplegia of childhood (50%), cerebellar ataxia (50%), and optic atrophy (23.08%). Non-classical symptoms included dystonia (73.08%), paroxysmal dyskinesias (34.62%), and encephalopathy (26.92%). Developmental delay was reported among 84.62% in cognitive, 92.31% in sensorimotor, 80.77% in speech, and 76.92% in socioemotional. EEG and MRI were non-specific. CONCLUSION: Our study demonstrated high heterogeneity among patients with pathogenic variants in the ATP1A3 gene. Such variation is multifactorial and can be a predisposition of wide genetic and clinical variables. Many patients shared few similarities in their genetic map including repeatedly reported de novo, heterozygous, mutations in the gene. Clinically, higher females prevalence of atypical presentation was noted. These findings are validated with prior evidence and the comprehensive analysis in this study.

VCIP135 associates with both the N- and C-terminal regions of p97 ATPase.

Two distinct p97ATPase-mediated membrane fusion pathways are required for Golgi and endoplasmic reticulum (ER) biogenesis, namely, the p97/p47 pathway and the p97/p37 pathway. p97 (VCP)/p47 complex-interacting protein p135 (VCIP135) is necessary for both of these pathways. Although VCIP135 is known to form a complex with p97 in the cytosol, the role of this complex in Golgi and ER biogenesis has remained unclear. In this study, we demonstrated that VCIP135 has two distinct p97-binding sites at its N- and C-terminal regions. In particular, the C-terminal binding site includes the SHP motif, which is also found in other p97-binding proteins, such as p47, p37, and Ufd1. We also clarified that VCIP135 binds to both the N- and C-terminal regions of p97; that is, the N- and C-terminal binding sites in VCIP135 interact with the C- and N-terminal regions of p97, respectively. These two interactions within the complex are synchronously controlled by the nucleotide state of p97. We next generated VCIP135 mutants lacking each of the p97-binding sites to investigate their functions in living cells and clarified that VCIP135 is involved in Golgi and ER biogenesis through its two distinct interactions with p97. VCIP135 is hence a unique p97-binding protein that functions by interacting with both the N-and C-terminal regions of p97, which strongly suggests that it plays crucial roles in p97-mediated events.

The fidgetin family: Shaking things up among the microtubule-severing enzymes.

The microtubule cytoskeleton is required for several crucial cellular processes, including chromosome segregation, cell polarity and orientation, and intracellular transport. These functions rely on microtubule stability and dynamics, which are regulated by microtubule-binding proteins (MTBPs). One such type of regulator is the microtubule-severing enzymes (MSEs), which are ATPases Associated with Diverse Cellular Activities (AAA+ ATPases). The most recently identified family are the fidgetins, which contain three members: fidgetin, fidgetin-like 1 (FL1), and fidgetin-like 2 (FL2). Of the three known MSE families, the fidgetins have the most diverse range of functions in the cell, spanning mitosis/meiosis, development, cell migration, DNA repair, and neuronal function. Furthermore, they offer intriguing novel therapeutic targets for cancer, cardiovascular disease, and wound healing. In the two decades since their first report, there has been great progress in our understanding of the fidgetins; however, there is still much left unknown about this unusual family. This review aims to consolidate the present body of knowledge of the fidgetin family of MSEs and to inspire deeper exploration into the fidgetins and the MSEs as a whole.

The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1.

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity.

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

Plant P4-ATPase lipid flippases: How are they regulated?

P4 ATPases are active membrane transporters that translocate lipids towards the cytosolic side of the biological membranes in eukaryotic cells. Due to their essential cellular functions, P4 ATPase activity is expected to be tightly controlled, but fundamental aspects of the regulation of plant P4 ATPases remain unstudied. In this mini-review, our knowledge of the regulatory mechanisms of yeast and mammalian P4 ATPases will be summarized, and sequence comparison and structural modelling will be used as a basis to discuss the putative regulation of the corresponding plant lipid transporters.

Regulation of the apico-basolateral trafficking polarity of the homologous copper-ATPases ATP7A and ATP7B.

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Primary architecture and energy requirements of Type III and Type IV secretion systems.

Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through a one-step mechanism. The secreted substrates (protein or nucleo-protein complexes in the case of Type IV conjugative systems) are guided to the base of the secretion channel, where they are directly delivered into the host cell in an ATP-dependent unfolded state. Despite the numerous disparities between these secretion systems, here we have focused on the structural and functional similarities between both systems. In particular, on the structural similarity shared by one of the main ATPases (EscN and VirD4 in Type III and Type IV secretion systems, respectively). Interestingly, these ATPases also exhibit a structural resemblance to F1-ATPases, which suggests a common mechanism for substrate secretion. The correlation between structure and function of essential components in both systems can provide significant insights into the molecular mechanisms involved. This approach is of great interest in the pursuit of identifying inhibitors that can effectively target these systems.

The Plasma Membrane H+ ATPase CsPMA2 Regulates Lipid Droplet Formation, Appressorial Development and Virulence in Colletotrichum siamense.

Plasma membrane H+-ATPases (PMAs) play an important role in the pathogenicity of pathogenic fungi. Lipid droplets are important storage sites for neutral lipids in fungal conidia and hyphae and can be used by plant pathogenic fungi for infection. However, the relationship between plasma membrane H+-ATPase, lipid droplets and virulence remains unclear. Here, we characterized a plasma membrane H+-ATPase, CsPMA2, that plays a key role in lipid droplet formation, appresorial development and virulence in C. siamense. Deletion of CsPMA2 impaired C. siamense conidial size, conidial germination, appressorial development and virulence but did not affect hyphal growth. ΔCsPMA2 increased the sensitivity of C. siamense to phytic acid and oxalic acid. CsPMA2 was localized to lipids on the plasma membrane and intracellular membrane. Deletion of CsPMA2 significantly inhibited the accumulation of lipid droplets and significantly affected the contents of some species of lipids, including 12 species with decreased lipid contents and 3 species with increased lipid contents. Furthermore, low pH can inhibit CsPMA2 expression and lipid droplet accumulation. Overall, our data revealed that the plasma membrane H+-ATPase CsPMA2 is involved in the regulation of lipid droplet formation and affects appressorial development and virulence in C. siamense.

Elucidating Events within the Black Box of Enzyme Catalysis in Energy Metabolism: Insights into the Molecular Mechanism of ATP Hydrolysis by F1-ATPase.

Oxygen exchange reactions occurring at ß-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F1-ATPase (MF1). The apparent rate constant of oxygen exchange governing the intermediate Pi-HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1-5000 µM) and a corresponding ~200-fold range of reaction velocity (3.5-650 [moles of Pi/{moles of F1-ATPase}-1 s-1]). Isotopomer distributions of [18O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F1-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F1-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F1-ATPase have been discussed. Current models of ATP hydrolysis by F1-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath's torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F1-ATPase.