62 research outputs found
The structural basis for pyrophosphatase catalysis
AbstractBackground Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable.Results We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 å resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions.Conclusions Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the ‘two-metal ion’ mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This ‘three-metal ion’ mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is
RETAK KOROSI TEGANGAN PADA PRESSURE INDICATOR LINE TUBE
RETAK KOROSI TEGANGAN PADA PRESSURE INDICATOR LINE TUBE. Pressure indicator line tubemengalami retak yang cukup dominan akibat mekanisme Intercrystalline Stress Corrosion Cracking (ISCC) pada bagianelbownya. Tube tersebut terbuat daTi stainless steel type 304 berdiameter luar 12,70 mm dengan ketebalan 3,60- 3,90 mmyang dioperasikan pada peralatan industri petrokimia. Pada daerah elbow tersebut mengandung residual surface tensile stresses.kemungkinan pada saat proses pembentukan elbow tidak diikuti perlakuan panas pelunakan (annealing) ataupun pelepasantegangan (stress relieving). Kondisi ini akan sangat sensitif ketika pressure indicator tube dioperasikan pada temperatur500-600°C adanya pemuaian panas (thermal expansion) menimbulkan tegangan tambahan (addition stress) Sedangkancorrosion agent yang mengawali terbentuknya stress corrosion cracking pada tube elbow adalah ion Cl- (berasal dan atmostirair laut) yang terperangkap di bawah glass wool insulation clan terkonsentrasi di daerah sisi cekung. Secara kuantitatif adanyategangan pada elbow sisi cekung dapat dilakukan perhitungan melalui pendekatan thermal stress analysis, sedangkan untukmenghindari kerusakan serupa dikemudian hari sebaiknya material pipa pressure indicator tube diganti dengan material yanglebih baik yaitu SS 316 atau SS 316L
Two independent evolutionary routes to Na+/H+ cotransport function in membrane pyrophosphatases.
Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi)
to transport H(+), Na(+) or both and help organisms to cope with stress
conditions, such as high salinity or limiting nutrients. Recent
elucidation of mPPase structure and identification of subfamilies that
have fully or partially switched from Na(+) to H(+) pumping have
established mPPases as versatile models for studying the principles
governing the mechanism, specificity and evolution of cation
transporters. In the present study, we constructed an accurate
phylogenetic map of the interface of Na(+)-transporting PPases
(Na(+)-PPases) and Na(+)- and H(+)-transporting PPases
(Na(+),H(+)-PPases), which guided our experimental exploration of the
variations in PPi hydrolysis and ion transport activities during
evolution. Surprisingly, we identified two mPPase lineages that
independently acquired physiologically significant Na(+) and H(+)
cotransport function. Na(+),H(+)-PPases of the first lineage transport
H(+) over an extended [Na(+)] range, but progressively lose H(+)
transport efficiency at high [Na(+)]. In contrast, H(+)-transport by
Na(+),H(+)-PPases of the second lineage is not inhibited by up to 100 mM
Na(+) With the identification of Na(+),H(+)-PPase subtypes, the mPPases
protein superfamily appears as a continuum, ranging from monospecific
Na(+) transporters to transporters with tunable levels of Na(+) and H(+)
cotransport and further to monospecific H(+) transporters. Our results
lend credence to the concept that Na(+) and H(+) are transported by
similar mechanisms, allowing the relative efficiencies of Na(+) and H(+)
transport to be modulated by minor changes in protein structure during
the course of adaptation to a changing environment. </p
Cystathionine beta-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria
Among numerous proteins containing pairs of regulatory cystathionine beta-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P-1,P-n-diadenosine 5'-polyphosphates (Ap(n)As, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap(4)A, Ap(5)A, and Ap(6)A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap(3)A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All Ap(n)As abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were similar to 10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap(4)A and Ap(5)A but 5.5 kcal/mol and 2 mol/mol for Ap(3)A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain noDRTGG domain, were not affected by Ap(n)As and showed no enthalpy change, indicating the importance of the DTRGG domain for Ap(n)A binding. These findings suggest that Ap(n)As can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.</p
Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na+-translocating pyrophosphatase
Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H+ pump that transports the H+ ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na+ via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [P-32]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na+, whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H2O with D2O decreased the rates of pyrophosphate hydrolysis by both Na+- and H+-transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na+-pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H+-pumping and Na+-pumping by mPPases
Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides
Inorganic pyrophosphatase containing regulatory cystathionine β-synthase
(CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP)
and adenosine diphosphate and activated by adenosine triphosphate (ATP)
and diadenosine polyphosphates; mononucleotide binding to CBS domains
and substrate binding to catalytic domains are characterized by positive
cooperativity. This behavior implies three pathways for regulatory
signal transduction — between regulatory and active sites, between two
active sites, and between two regulatory sites. Bioinformatics analysis
pinpointed six charged or polar amino acid residues of Desulfitobacterium hafniense
CBS-PPase as potentially important for enzyme regulation. Twelve mutant
enzyme forms were produced, and their kinetics of pyrophosphate
hydrolysis was measured in wide concentration ranges of the substrate
and various adenine nucleotides. The parameters derived from this
analysis included catalytic activity, Michaelis constants for two active
sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for
two regulatory sites, and the degree of activation/inhibition for each
nucleotide. Replacements of arginine 295 and asparagine 312 by alanine
converted ATP from an activator to an inhibitor and markedly affected
practically all the above parameters, indicating involvement of these
residues in all the three regulatory signaling pathways. Replacements of
asparagine 312 and arginine 334 abolished or reversed kinetic
cooperativity in the absence of nucleotides but conferred it in the
presence of diadenosine tetraphosphate, without effects on
nucleotide-binding parameters. Modeling and molecular dynamics
simulations revealed destabilization of the subunit interface as a
result of asparagine 312 and arginine 334 replacements by alanine,
explaining abolishment of kinetic cooperativity. These findings identify
residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS
domains.</p
A Monte Carlo method to estimate the confidence intervals for the concentration index using aggregated population register data
New genetic loci link adipose and insulin biology to body fat distribution.
Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms
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