Alexander A. Baykov

Evolutionary conservation of the active site of soluble inorganic pyrophosphatase

Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.

Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes

An open reading frame located in the COTF‐TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+‐activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

The CBS domain: a protein module with an emerging prominent role in regulation.

Regulatory CBS (cystathionine β-synthase) domains exist as two or four tandem copies in thousands of cytosolic and membrane-associated proteins from all kingdoms of life. Mutations in the CBS domains of human enzymes and membrane channels are associated with an array of hereditary diseases. Four CBS domains encoded within a single polypeptide or two identical polypeptides (each having a pair of CBS domains at the subunit interface) form a highly conserved disk-like structure. CBS domains act as autoinhibitory regulatory units in some proteins and activate or further inhibit protein function upon binding to adenosine nucleotides (AMP, ADP, ATP, S-adenosyl methionine, NAD, diadenosine polyphosphates). As a result of the differential effects of the nucleotides, CBS domain-containing proteins can sense cell energy levels. Significant conformational changes are induced in CBS domains by bound ligands, highlighting the structural basis for their effects.

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

Evolutionary aspects of inorganic pyrophosphatase

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Cytoplasmic Inorganic Pyrophosphatase

Pyrophosphate (PPi) is the smallest member of the polyphosphate family and is formed by two phosphate (Pi) residues linked by a phosphoanhydride bond. A specific enzyme hydrolyzing PPi to Pi was discovered in animal tissues in 1928 (Kay) and later in a great many other organisms and cell types, in virtually all in which it has been sought. Its initial name was “pyrophosphatase”, later elongated with a questionable “inorganic”.

Quaternary Structure and Metal Ion Requirement of Family II Pyrophosphatases from Bacillus subtilis,Streptococcus gordonii, and Streptococcus mutans

Pyrophosphatase (PPase) from Bacillus subtilis has recently been found to be the first example of a family II soluble PPase with a unique requirement for Mn2+. In the present work, we cloned and overexpressed in Escherichia coli putative genes for two more family II PPases (fromStreptococcus mutans and Streptococcus gordonii), isolated the recombinant proteins, and showed them to be highly specific and active PPases (catalytic constants of 1700–3300 s− 1 at 25 °C in comparison with 200–400 s− 1 for family I). All three family II PPases were found to be dimeric manganese metalloenzymes, dissociating into much less active monomers upon removal of Mn2+. The dimers were found to have one high affinity manganese-specific site (K d of 0.2–3 nm for Mn2+and 10–80 μm for Mg2+) and two or three moderate affinity sites (K d ∼ 1 mmfor both cations) per subunit. Mn2+ binding to the high affinity site, which occurs with a half-time of less than 10 s at 1.5 mm Mn2+, dramatically shifts the monomer ↔ dimer equilibrium in the direction of the dimer, further activates the dimer, and allows substantial activity (60–180 s− 1) against calcium pyrophosphate, a potent inhibitor of family I PPases.

Differential sensitivity of membrane‐associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme

1,1‐Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H+‐translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 μM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H+‐pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

A Trimetal Site and Substrate Distortion in a Family II Inorganic Pyrophosphatase

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. Structures of both the fluoride-inhibited wild type and the H98Q variant of the PNP-Bacillus subtilis pyrophosphatase complex show a unique trinuclear metal center. Each metal ion coordinates a terminal oxygen on the electrophilic phosphate and a lone pair on the putative nucleophile, thus placing it in line with the scissile bond without any coordination by protein. The nucleophile moves further away from the electrophilic phosphorus site, to the opposite side of the trimetal plane, upon binding of substrate. In comparison with earlier product complexes, the side chain of Lys296 has swung in and so three positively charged side chains, His98, Lys205 and Lys296, now surround the bridging nitrogen in PNP. Finally, one of the active sites in the wild-type structure appears to show evidence of substrate distortion. Binding to the enzyme may thus strain the substrate and thus enhance the catalytic rate.