-galactosidase-catalysed hydrolysis of -D-galactopyranosyl azide.
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ABSTRACT:
SUBMITTER: Sinnott ML
PROVIDER: S-EPMC1178175 | biostudies-other | 1971 Dec
REPOSITORIES: biostudies-other
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Project description:The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).
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Project description:The genus of Termitomyces purchased from the market has been identified as Termitomyces eurrhizus using the Internal Transcribed Spacer (ITS) method. An α-galactosidase from T. eurrhizus (TEG), a monomeric protein with a molecular mass of 72 kDa, was purified 146 fold by employing ion exchange chromatography and gel filtration. The optimum pH and temperature was 5.0 and 60 °C, respectively. TEG was stable over pH 2-6, and also exhibited good thermostablility, retaining 100% of the original activity after incubation at 60 °C for 2 h. Inhibition of the enzyme activity by N-bromosuccinimide (NBS) constituted evidence for an essential role of tryptophan in the catalytic action of the isolated enzyme. Besides 4-nitro-phenyl α-d-galactophyranoside (pNPGal), natural substrates could also be effectively hydrolyzed by TEG. Results of thin-layer chromatography (TLC) revealed complete enzymatic hydrolysis of raffinose and stachyose to galactose at 50 °C within 6 h. These properties of TEG advocate its utilization for elevating the nutritional value of soymilk.
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Project description:Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.
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Project description:1. The Michaelis-Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k(0), is 2-3sec.(-1) and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k(0), less than 2-3sec.(-1). It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme-substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis-Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, sigma. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-alpha-amino acid derivatives.
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Project description:1. The activated amide (4-nitroanilide), N-(4-nitrophenyl) N'-butyl-1,4-phenylenediacetamide (III) was synthesized. 2. A polyclonal antibody preparation (PCA 270-29) was elicited in a multigeneration cross-bred sheep (no. 270) and isolated 29 weeks into the immunization schedule by procedures described previously for PCA 270-22 [Gallacher, Jackson, Searcey, Badman, Goel, Topham, Mellor & Brocklehurst (1991) Biochem J. 271, 871-881]. These involved the use of an amide conjugate bonded through the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin as the immunogen. 3. PCA 270-29 was shown to catalyse the hydrolysis of both the carbonate ester substrate 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I) and the amide substrate (III). Both catalyses obeyed the Michaelis-Menten equation with the following values of the parameters at 25 degrees C: for the hydrolysis of (I) at pH 8.0, Km = 3.96 +/- 0.28 microM and k(cat.) = 0.135 +/- 0.004 s-1 (k(non-cat.) = 1.99 x 10(-4) s-1); for the hydrolysis of (III) at pH 9.0, Km = 5.4 +/- 1.4 microM and k(cat.) = (5.95 +/- 0.75) x 10(-5) s-1 (k(non-cat.) = approx. 2 x 10(-7) s-1). 4. The finding that PCA 270-29 is almost equally effective as a catalyst for the hydrolysis of the amide (III) as for that of the carbonate ester (I) when allowance is made for the different intrinsic reactivities of the two types of substrate is discussed. The catalytic characteristics of PCA 270-29, the first example of a polyclonal catalytic antibody preparation shown to catalyse the hydrolysis of an amide and the first example of an antibody preparation (monoclonal or polyclonal) with such catalytic character to be produced by use of a phosphate immunogen, are compared with those of the small number of other antibody-mediated hydrolyses of amides in the literature.
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Project description:A novel intracellular ?-galactosidases produced by Lactobacillus plantarum HF571129, isolated from an Indian traditional fermented milk product curd was purified and characterized. The ?-galactosidases is a hetrodimer with a molecular weight of 60 kDa (larger subunit) and 42 kDa (smaller subunit), as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 7.23 fold by ultrasonication, ultrafiltration and gel filtration chromatography with an overall recovery of 30.41 %. The optimum temperature for hydrolysis of its preferred substrates, o-nitrophenyl- ?-D-galactopyranoside (ONPG) and lactose, are 50 °C (both), and optimum pH for these reactions is 6.5 and 7.5, respectively. The ?-galactosidases showed higher affinity for ONPG (Km, 6.644 mM) as compared to lactose (Km, 23.28 mM). Galactose, the end product of lactose hydrolysis was found to be inhibited (47 %). The enzyme activity was drastically altered by the metal ion chelators EDTA, representing that this enzyme is a metalloenzyme. The enzyme was activated to a larger extent by Mg(2+) (73 % at 1 mM), while inhibited at higher concentrations of Na(+) (54 % at 100 mM), K(+) (16 % at 100 mM) and urea (16 % at 100 mM). The thermal stability study indicated an inactivation energy of Ed?=?171.37 kJ mol(-1). Thermodynamic parameters such as ?H, ?S and ?G, were determined as a function of temperature. About 88 % of lactose was hydrolyzed at room temperature within 1 h. The study suggested that this enzyme showed its obvious superiority in the industrial lactose conversion process.
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