E. V. Rodina

Escherichia coli inorganic pyrophosphatase : site-directed mutagenesis of the metal binding sites

Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine. Each mutation was found to decrease the κ app value by approx. 2–3 orders of magnitude. At the same time, the K m values changed only slightly. Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis. All mutant variants have practically the same affinity to Mg2+ as the wild‐type pyrophosphatase.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4(2-). Ligand-induced molecular asymmetry.

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding

Asp‐42 located in the active site of E. coli inorganic pyrophosphatase (PPase) has been substituted by Asn by site‐directed mutagenesis. This resulted in a 3‐fold increase in hydrolytic activity measured under optimal conditions, a 15.5‐fold increase in the K m value and retention of the pK values of groups for enzyme and enzyme‐substrate complex. The active site of the enzyme contains 4 metal binding centers (I–IV) [Harutyunyan et al. (1996) Eur. J. Biochem., in press]. Asp‐42 is located near centers II and IV. The D42N replacement had no effect on Mg2+ binding with center II. At the same time, occupation of center IV eliminates the inhibition of inorganic pyrophosphate hydrolysis by high Mg2+ concentrations typical of wild‐type PPase. It is proposed that the increase in activity and decrease in affinity for substrate of the D42N PPase results from changes in Mg2+ binding to center IV. The Mg2+ binding centers of E. coli PPase are lined up in filling order.

Mg2+ activation of Escherichia coli inorganic pyrophosphatase

Further refinement of X‐ray data on Escherichia coli inorganic pyrophosphatase [Oganessyan et al. (1994) FEBS Lett. 348, 301–304] to 2.2 Å reveals a system of noncovalent interactions involving Tyr55 and Tyr141 in the active site. The pKa for one of the eight Tyr residues in wild‐type pyrophosphatase is as low as 9.1 and further decreases to 8.1 upon Mg2+ binding, generating characteristic changes in the absorption spectrum. These effects are lost in a Y55F but not in a Y141F variant. It is suggested that the lower‐affinity site for Mg2+ in the enzyme is formed by Tyr55 and Asp70, which are in close proximity in the apo‐enzyme structure.

Effectory Site in Escherichia coli Inorganic Pyrophosphatase is Revealed Upon Mutation at the Intertrimeric Interface

Escherichia coli inorganic pyrophosphatase (E‐PPase) is a homohexamer formed from two trimers related by a two‐fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E‐PPase is found to not obey Michaelis‐Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite. Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPPi hydrolysis though its magnesium complex is a competitive inhibitor. The Asp26Ala variant is the first example of hexameric E‐PPase demonstrated to have an activatory subsite. IUBMB Life, 55: 37‐41, 2003

Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase

Soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) is a hexamer forming under acidic conditions the active trimers. We have earlier found that the hydrolysis of a substrate (MgPPi) by the trimers as well as a mutant E-PPase Asp26Ala did not obey the Michaelis-Menten equation. To explain this fact, a model has been proposed implying the existence of, aside from an active site, an effector site that can bind PPi and thus accelerate MgPPi hydrolysis. In this paper, we demonstrate that the noncompetitive activation of MgPPi hydrolysis by metal-free PPi can also explain kinetic features of hexameric forms of both the native enzyme and the specially obtained mutant E-PPase with a substituted residue Glu145 in a flexible loop 144-149. Aside from PPi, its non-hydrolyzable analog methylene diphosphonate can also occupy the effector site resulting in the acceleration of the substrate hydrolysis. Our finding that two moles of [32P]PPi can bind with each enzyme subunit is direct evidence for the existence of the effector site in the native E-PPase.

Metal cofactors play a dual role in Mycobacterium tuberculosis inorganic pyrophosphatase.

Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators—Mg2+ and Mn2+. Dissociation constants of Mt-PPase complexed with Mg2+ or Mn2+ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg2+-or Mn2+-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg2+ or Mn2+ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8–10 toward the hexameric form by stabilizing intertrimeric contacts. The pKa values have been determined for groups that control the observed hexamer-monomer (pKa 5.4), hexamer-trimer (pKa 7.5), and trimer-monomer (pKa 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4 2

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Pattersons search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.

Nanomaterials based on peptides

Owing to the assortment of functional groups, peptides are capable of self-assembly and formation of regularly patterned supramolecular complexes. Peptide-based nanomaterials offer promise for medicine, engineering, and bioimaging. The present review surveys the structure and characteristics of filaments formed by peptides of various secondary structures and their further assembly into 2D and 3D nanomaterials. Possible application areas of self-assembling peptide systems, including the synthesis of inorganic nanomaterials, are considered.

ATP as effector of inorganic pyrophosphatase of Escherichia coli. Identification of the binding site for ATP

The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors.