Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

Brandão, Tiago A. S.; Robinson, Howard; Johnson, Sean; Hengge, Alvan C.

doi:10.1021/ja807418b

Cited by 44 publications

(72 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In comparison with YopH, there are 119 crystal structures of proteins bound to PP i available in the PDB. The structure of YopH demonstrates how a mutation in the hinge region impairs catalytic activity and reveals a unique transition state-like structure of VV (18).…”

Section: Discussionmentioning

confidence: 98%

“…The two charged vanadate moieties occupy the binding loci for the carboxylate and phosphate of the monophosphoglycerate in the active site of the enzyme. The only crystal structure available for an enzyme bound to VV is of YopH, a tyrosine phosphatase produced by a pathogenic Yersinia species (18). In comparison with YopH, there are 119 crystal structures of proteins bound to PP i available in the PDB.…”

Section: Discussionmentioning

confidence: 99%

“…Binding of VV to phosphoglycerate mutase was found to activate the enzyme and the transfer of phosphate directly to a water molecule (17). A crystal structure of VV bound in the active site of the tyrosine phosphatase YopH has been reported recently (18). To our knowledge, these examples are among the few structural and biochemical evidences for the binding and activation of an enzymatic reaction by VV.…”

mentioning

confidence: 95%

See 2 more Smart Citations

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Akabayov¹,

Kulczyk²,

Akabayov³

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

DNA polymerases catalyze the 3-5-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP i ). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP i , a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP i complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn 2؉ , larger than Mg 2؉ , fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for replication of the T7 genome (1). T7 DNA polymerase forms a complex with Escherichia coli thioredoxin (trx), 2 an interaction that increases its processivity of nucleotide polymerization several hundredfold (2). The structure of T7 DNA polymerase in complex with trx is shown in Fig. 1. The structure closely resembles that of E. coli DNA polymerase I, thus placing it in the polymerase I family of DNA polymerases (3). In this study, we designate the polymerase in complex with trx as T7 DNA polymerase. Like most other prokaryotic DNA polymerases, T7 DNA polymerase has a proofreading 3Ј-5Ј-exonuclease activity located in the amino-terminal half of the protein (3). The crystal structure shows that its active site is located between the "fingers" and the "palm" subdomains and contains two magnesium ions (Mg 2ϩ ). During polymerization of nucleotides, the Mg 2ϩ closest to the primer (catalytic Mg 2ϩ ) is responsible for deprotonation of the 3Ј-hydroxyl group of the primer prior to its nucleophilic attack on the ␣-phosphate of the incoming dNTP. The second Mg 2ϩ (structural Mg 2ϩ ) is associated with stabilization of the reactive state of the ␤-and ␥-phosphates of the incoming deoxynucleoside 5Ј-triphosphate (dNTP) (4).In the presence of PP i , DNA polymerases catalyze the 3Ј-5Ј-degradation of a DNA primer annealed to a DNA template (5). In this reaction, known as pyrophosphorolysis, the deoxynucleotides (dNMP) of the DNA are converted to dNTP. Pyrophosphorolysis is a true reversal of the polymerization reaction in that the products are dNTPs, and it has the same requirements for a template and a 3Ј-hydroxyl-terminated primer (5). The 3Ј-5Ј-exonuclease associated with DNA polymerase, however, catalyzes the 3Ј-5Ј hydrolysis of the primer to yield dNMPs without a strict requirement for proper base pairing. Pyrophosphorolysis o...

show abstract

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

See 1 more Smart Citation

Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

Akabayov¹,

Kulczyk²,

Akabayov³

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Vanadate anions form stable tetrahedral structures in solution but exhibit clear preference for trigonal bipyramidal structures in the presence of polydentate ligands (65), including the active site of enzymes, such as PTPs (20,22). In these enzymes, vanadate exhibits K i values ranging from 0.4 to 5 M (23, 68, 69) and structural characteristics resembling the trigonal bipyramidal transition state in phosphoryl transfer reactions.…”

Section: The Mechanism Of Catalysis By Protein-tyrosine Phosphatase 1bmentioning

confidence: 99%

Insights into the Reaction of Protein-tyrosine Phosphatase 1B

Brandão

Hengge

Johnson

2010

Journal of Biological Chemistry

Self Cite

132

175

View full text Add to dashboard Cite

Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O-O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.The phosphorylation of tyrosine residues by protein-tyrosine kinases and the reverse action by protein-tyrosine phosphatases (PTPs) 4 is a common mechanism for the control of biological pathways (1-3). Protein-tyrosine phosphatase 1B (PTP1B) is a biomedically important phosphatase with several roles, including negative regulation of insulin signaling by dephosphorylation of the insulin receptor tyrosine kinase (4). Knock-out studies show that loss of PTP1B is associated with an increased insulin sensitivity and suppression of weight gain in mice (5). As a result, this enzyme has been considered a significant target for treatment of type 2 diabetes and obesity (6, 7). PTP1B also down-regulates cell growth by dephosphorylating the epidermal growth factor receptor (8). Overexpression has been observed in human breast and ovarian cancer, where it is believed to suppress potential tumors by antagonizing signaling of oncogenic factors (9, 10). Other PTPs are known virulence factors for a number of human diseases (11-13).Reactions catalyzed by PTPs take place by a ping-pong mechanism ( Fig. 1) (2). In the first step, a nucleophilic cysteine thiolate attacks the phosphate ester moiety of the substrate, resulting in formation of a phosphoenzyme intermediate with release of the peptidyl tyrosine. The second step occurs via attack of water on the phosphoenzyme intermediate and yields the final products inorganic phosphate and the regenerated enzyme. The central binding site for the substrate is the P-loop, a region at the bottom of a pocket that includes the nucleophilic cysteine and backbone amide groups oriented in a horseshoe fashion. The amide protons in the P-loop, together with a conserved arginine residue, hydrogen-bond to the phosphoryl group of the substrate and orient it for nucleophilic attack, providing transition state (TS) stabilization. Substrate binding is followed by conformational changes that culminate with closure of the activ...

show abstract

“…The same authors reported the binding of a divanadate complex to a protein molecule. In the structure of the Yersinia PTP YopH, the oxoanion exhibits a double distorted trigonal bipyramid containing a cyclic [VO] 2 core[257].…”

mentioning

confidence: 99%