Participation of Two Carboxyl Groups in Phosphodiester Hydrolysis. 1. Hydrolysis of Bis(2-carboxyphenyl) Phosphate

1995 ◽  
Vol 117 (49) ◽  
pp. 12064-12069 ◽  
Author(s):  
Thomas C. Bruice ◽  
Andrei Blasko ◽  
Mark E. Petyak
Keyword(s):  
Carboxyl Groups ◽  
Phosphodiester Hydrolysis ◽  
Hydrolysis Of

scholarly journals Pseudomonas putida MPE, a manganese-dependent endonuclease of the binuclear metallophosphoesterase superfamily, incises single-strand DNA in two orientations to yield a mixture of 3′-PO4 and 3′-OH termini

2020 ◽  
Author(s):  
Shreya Ghosh ◽  
Anam Ejaz ◽  
Lucas Repeta ◽  
Stewart Shuman
Keyword(s):  
Pseudomonas Putida ◽  
Bound Water ◽  
Acid Catalyst ◽  
Nuclease Activity ◽  
Single Strand ◽  
Dna Endonuclease ◽  
Dependent Endonuclease ◽  
Leaving Group ◽  
Phosphodiester Hydrolysis ◽  
Hydrolysis Of

Abstract Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3′ and 5′ single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3′-OH and 3′-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.

THE PROBLEM OF PLASTEIN FORMATION: II. THE CHEMICAL CHANGES INVOLVED IN PLASTEIN FORMATION BY PAPAIN AND BY PEPSIN

1940 ◽  
Vol 18b (9) ◽  
pp. 272-280 ◽  
Author(s):  
H. B. Collier
Keyword(s):  
Enzymatic Hydrolysis ◽  
Free Amino ◽  
Essential Role ◽  
Phenolic Hydroxyl ◽  
Carboxyl Groups ◽  
Hydroxyl Groups ◽  
Chemical Changes ◽  
Phenolic Hydroxyl Groups ◽  
Hydrolysis Of

It has been confirmed that free amino and carboxyl groups disappear during plastein formation from concentrated proteose by crystalline pepsin. Using papain, the changes are obscured by simultaneous hydrolysis. Enzymatic hydrolysis of the plasteins results in the liberation of free amino and carboxyl groups.Reactive "tyrosine" decreases during plastein formation by either enzyme. The same groups are liberated on enzymatic hydrolysis of the plasteins, in a manner analogous to that which takes place in the hydrolysis of typical proteins.It is concluded that in so far as the changes in amino, carboxyl, and "tyrosine" groups are concerned, the plasteins are similar to typical proteins. It is further suggested that the phenolic hydroxyl groups of tyrosine play an essential role in the structure of the protein molecule.Benzaldehyde was found to have no effect on the formation of plastein from proteose by crystalline pepsin.

Anchimeric assistance in hexosaminidases

2002 ◽  
Vol 80 (8) ◽  
pp. 1064-1074 ◽  
Author(s):  
Brian L Mark ◽  
Michael NG James
Keyword(s):  
Oxygen Atom ◽  
Active Site ◽  
Sequence Similarity ◽  
Carbonyl Oxygen ◽  
Carbonyl Oxygen Atom ◽  
Carboxyl Groups ◽  
Displacement Mechanism ◽  
Glycosidic Bonds ◽  
Anchimeric Assistance ◽  
Hydrolysis Of

Configuration retaining glycosidases catalyse the hydrolysis of glycosidic bonds via a double displacement mechanism, typically involving two key active site carboxyl groups (Glu or Asp). One of the enzymic carboxyl groups functions as a general acid–base catalyst, the other acts as a nucleophile. Alternatively, configuration-retaining hexosaminidases from the sequence-related glycosidase families 18, 20, and 56 lack a suitably positioned enzymic nucleophile; instead, they use the carbonyl oxygen atom of the neighbouring C2-acetamido group of the substrate. The carbonyl oxygen atom of the 2-acetamido group provides anchimeric assistance to the enzyme catalyzed reaction by acting as an intramolecular nucleophile, attacking the anomeric center and forming a cyclized oxazolinium ion intermediate that is stereochemically equivalent to the glycosyl–enzyme intermediate formed in the "normal" double displacement mechanism. Although there is little sequence similarity between families 18, 20, and 56 hexosaminidases, X-ray crystallographic studies demonstrate that they have evolved similar catalytic domains and active site architectures that are designed to distort the bound substrate so that the C2-acetamido group can become appropriately positioned to participate in catalysis. The substrate distortion allows for a substrate-assisted catalytic reaction that displays all the general characteristics of the classic double-displacement mechanism including the formation of a covalent intermediate.Key words: glycoside hydrolase, hexosaminidase, glycosidase, substrate-assisted catalysis, anchimeric assistance.

Immobilisierung von Cytidin und Uridin über 2'.3'-O-cyclische Acetalderivate an Agarose / Im mobilisation of Cytidine and Uridine via 2',3'-O-Cyclic Acetal Derivatives to Agarose

1978 ◽  
Vol 33 (1-2) ◽  
pp. 56-60 ◽  
Author(s):  
Frank Seela ◽  
Helmut Roseineyer
Keyword(s):  
Nmr Spectroscopy ◽  
Absolute Configuration ◽  
Alkaline Hydrolysis ◽  
Carboxyl Groups ◽  
Cyclic Acetal ◽  
Ethyl Group ◽  
Ethyl Levulinate ◽  
The Absolute ◽  
Hydrolysis Of ◽  
C Nmr

Abstract Condensation of cytidine or uridine with ethyl levulinate leads to the acetals 1a/2a. The reac­tion would be expected to give mixtures of diastereoisom ers. As shown by 1H and 13C NMR spectroscopy only one diastereoisomer is formed. By spectroscopic comparison of 1a/2a with the corresponding adenosine acetal the absolute configuration of the new chiral centre was found to be R. The acetal m ethyl group of 1a/2a in exo-location can serve to distinguish the two m ethyl signals of O-2′,3′-isopropylidenecytidine and -uridine in the NM R spectra. On alkaline hydrolysis of the esters the acids 1b and 2b are formed, which can be condensed through their carboxyl groups with 6-aminohexylagarose. The affinity resins 3 and 4 contain 7.1 μmol and 7.6 μmol ligand/g moist gel respectively. A biospecificity of the new polymers to cytidine-and uridine converting enzymes is expected.

scholarly journals Chemically modified nylons as supports for enzyme immobilization. Polyisonitrile-nylon

1974 ◽  
Vol 143 (3) ◽  
pp. 497-509 ◽  
Author(s):  
Leon Goldstein ◽  
Amihay Freeman ◽  
Mordechai Sokolovsky
Keyword(s):  
Aqueous Medium ◽  
Enzyme Immobilization ◽  
Functional Group ◽  
Carboxyl Groups ◽  
Partial Hydrolysis ◽  
Amino Groups ◽  
Tyrosine Residues ◽  
Chemically Modified ◽  
Reactive Groups ◽  
Hydrolysis Of

Four-component condensations between amine, carboxyl, isocyanide and aldehyde lead to the formation of N-substituted amides (Ugi, 1962). The present paper describes the use of such condensations for the introduction of chemically reactive groups on to the polyamide backbone of nylon. Polyisonitrile-nylon was synthesized by partial hydrolysis of nylon-6 powder, followed by resealing of the newly formed −CO2... NH2− pairs via a four-component condensation, by using acetaldehyde and 1,6-di-isocyanohexane. Polyisonitrile-nylon could also be converted into a diazotizable arylamino derivative, polyaminoaryl-nylon, by a four-component condensation by using a bifunctional amine, pp′-diaminodiphenylmethane, in the presence of an aldehyde and a carboxylate compound. The versatility of four-component condensations involving the isocyanide functional group of polyisonitrile-nylon allowed coupling of proteins, in an aqueous medium at neutral pH, through either their amino or carboxyl groups. Trypsin and papain were bound to polyisonitrile-nylon through their amino groups by a four-component condensation by using acetaldehyde and acetate; conversely, succinyl-(3-carboxypropionyl-)trypsin, pepsin and papain were coupled through their carboxyl groups in the presence of acetaldehyde and an amine (Tris). Diazotized polyaminoaryl-nylon could be utilized for the immobilization of papain, via the tyrosine residues of the enzyme.

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