A new synthesis of benzoyl phosphate: a substrate for acyl phosphatase assay.
TL;DR: A new method for the synthesis of benzoyl phosphate was reported, which has the advantages of more rapid procedure, lower cost, and higher yield.
Abstract: A new method for the synthesis of benzoyl phosphate was reported. The advantages are: 1. more rapid procedure; 2. lower cost; 3. higher yield.
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TL;DR: The application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity is reported, based on the optimization of the energy of charge–charge interactions on the protein surface.
Abstract: Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge–charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.
207 citations
Cites methods from "A new synthesis of benzoyl phosphat..."
...How does this increase in stability affect the enzymatic activity of AcPh? The activity of AcPh proteins was compared by using synthetic substrate benzoylphosphate (35, 36)....
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TL;DR: It is determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility, and alternative pathways that bypass the PGK step of glycolysis exist.
Abstract: Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.
197 citations
TL;DR: In this article, the aggregation of α/β protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium.
134 citations
TL;DR: Phosphoenzyme-trapping experiments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate.
Abstract: Low-Mr phosphotyrosine protein phosphatase (PTPase), previously known as low-Mr acid phosphatase, catalyzes the in-vitro hydrolysis of tyrosine phosphorylated proteins, low-Mr aryl phosphates and natural and synthetic acyl phosphates. Its activity on Ser/Thr-phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl-phosphate hydrolysis was studied by means of non-mutated and mutated PTPase fusion proteins. The mechanism of benzoyl-phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme-catalyzed mechanisms. Nevertheless, the performed phospho-enzyme-trapping experiments enable us to identify Cys12 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cys17 in the substrate binding was confirmed, its participation a second time in the step that involves the Cys12 dephosphorylation was suggested by the results of phosphoenzyme-trapping experiments. The participation of Arg18 in the substrate-binding site was demonstrated by site-directed mutagenesis that produced the conservative Lys18 and the non-conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme-trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.
101 citations
TL;DR: Results suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state, which has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.
96 citations
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TL;DR: In this article, an investigation was carried out on a method for the determination of inorganic phosphate which is applicable to the study of phosphate-splitting enzymes and showed that after the color reagent has been added any inorganic phosphoric acid formed cannot react with molybdenum, for the latter has been complexed by the addition of a citrate-arsenite solution.
367 citations
167 citations
TL;DR: In this article, a metodo ottico continuo for determinazione dell'acilfosfatasi, usando come substrato il benzoilfosfato di litio, is described.
Abstract: Viene descritto un metodo ottico continuo per la determinazione dell'acilfosfatasi, usando come substrato il benzoilfosfato di litio. II metodo si basa sulla differenza di estinzione tra benzoilfosfato e benzoato.
67 citations
TL;DR: Two aromatic acyl phosphates were synthesized and their suitable optical characteristics, particularly of benzoyl phosphate, make it possible to use them as advantageous substrates for the continuous measurement of enzyme activity.
37 citations