The structural basis for pyrophosphatase catalysis

Heikinheimo, Pirkko; Lehtonen, Jukka V.; Baykov, Alexander A.; Lahti, Reijo; Cooperman, Barry S.; Goldman, Adrian

doi:10.1016/s0969-2126(96)00155-4

Cited by 137 publications

(231 citation statements)

References 62 publications

Supporting

Mentioning

212

Contrasting

Order By: Relevance

“…The pK a 6.3 in our case may correspond to a histidine residue as the pK a of free histidine in water is 6.3. Metalbridging hydroxide ion has been found to act as a nucleophile in a number hydrolytic manganese metalloenzymes such as inorganic pyrophosphatase (33), serine/threonine phosphatase-1 (34) and proline specific aminopeptidase from E. coli (35). Our data show that there is a variation in the pH dependence of the H. pylori enzyme with the metal ions; Co 21 showed a single ionization, whereas Mn 21 showed double ionization.…”

Section: Ph-dependent Studiesmentioning

confidence: 69%

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Srivastava

2010

IUBMB Life

View full text Add to dashboard Cite

SummaryArginase is a binuclear Mn 21 -metalloenzyme of urea cycle that catalyzes the conversion of L-arginine to L-ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co 21 , and has lower catalytic activity. To understand the differences in the biochemical properties as well as activity compared to other arginases, we carried out a detailed investigation of different metal reconstituted H. pylori arginases that includes steady-state kinetics, fluorescence measurement, pH-dependent and oligomerization assays. Unlike other arginases (except human at physiological pH), the Co 21 -and Mn 21 -reconstituted H. pylori enzymes exhibit cooperative mechanism of arginine hydrolysis, and undergo self-association and activation with increasing concentrations. Analytical gel-filtration assays in conjunction with the kinetic data showed that the protein exists as a mixture of monomer and dimer with monomer being the major form (other arginases exclusively exist as a trimer or hexamer) but the dimer is associated with higher catalytic activity. The proportion of dimer is found to decrease with increasing salt concentrations indicating that salt bridges play important roles in dimerization of the protein. Furthermore, the fluorescence measurement showed that Co 21 ions play an important role in the local tertiary structure of the protein than Mn 21 . This is consistent with the pH-dependent studies where the Co 21 -enzyme showed a single ionization compared to the double in the Mn 21 -enzyme. Thus, this study presents the detailed biochemical and spectroscopic investigations into the differences in the biochemical properties and activity between H. pylori and other arginases.

show abstract

Section: Ph-dependent Studiesmentioning

confidence: 69%

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Srivastava

2010

IUBMB Life

View full text Add to dashboard Cite

show abstract

“…In contrast, Glu 197 and Glu 202 may be directly involved in metal cofactor binding. According to Scheme I, the enzyme has a metal-binding site (M1) to form EM and an additional metalbinding site (M2) to bind M 2 PP if one of the two metal ions that come with PP i bridges PP i and the enzyme in the resulting EM 3 PP complex as in the case of soluble PPases (32,33). Glu 197 may be part of the M1 site as its substitution markedly increases K M1 , but not K A1 and K A2 (Table II), whereas Glu 202 possibly belongs to both the M1 site and the M2 site because its substitution increases all of these parameters.…”

Section: Discussionmentioning

confidence: 99%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

H ؉ -pyrophosphatase (H ؉ -PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H ؉ -PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu 197 , Glu 202 , Glu 550 , and Glu 649 in the H ؉ -PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP i hydrolysis and PP i -energized proton translocation, indicating that the negative charge of Glu 202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg 2؉ concentrations but were only slightly affected at high Mg 2؉ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two-and three-metal pathways. Substitution of Glu 197 , Glu 202 , and Glu 649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu 550 and Glu 649 , R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP i hydrolysis to proton transport.

show abstract

“…This cleft is used by various proteins to engage RNA (61), DNA (46), oligosaccharides (62), proteins (63), and even metals and inorganic phosphates (64,65). For example, RPA, a eukaryotic recruiting and scaffolding protein critical to DNA replication, has been shown to bind both oligonucleotides and peptides through its six OB-fold domains (66 -70).…”

Section: Chemical Nature Of Mcm10-id Interactions With Dna Andmentioning

confidence: 99%

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Warren

Huang²,

Fanning³

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

show abstract

The structural basis for pyrophosphatase catalysis

Cited by 137 publications

References 62 publications

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Contact Info

Product

Resources

About