ABSTRACT
beta-Aspartyl di- and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of beta-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of beta-aspartyl peptides by transferring the beta-aspartyl moiety from other beta-aspartyl peptides or beta-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the beta-aspartyl donor in the formation of beta-aspartyl peptides. The data show that synthesis and degradation of beta-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of beta-aspartyl peptides.
Subject(s)
Aspartic Acid/chemistry , Aspartylglucosylaminase/metabolism , Oligopeptides/biosynthesis , Catalysis , Hydrolysis , Oligopeptides/chemistry , Oligopeptides/metabolismABSTRACT
Glycosylasparaginase is a lysosomal amidase involved in the degradation of glycoproteins. Recombinant human glycosylasparaginase is capable of catalyzing the hydrolysis of the amino acid L-asparagine to L-aspartic acid and ammonia. For the hydrolysis of L-asparagine the Km is 3-4-fold higher and Vmax 1/5 of that for glycoasparagines suggesting that the full catalytic potential of glycosylasparaginase is not used in the hydrolysis of the free amino acid. L-Asparagine competitively inhibits the hydrolysis of aspartylglucosamine indicating that both the amino acid and glycoasparagine are interacting with the same active site of the enzyme. The hydrolytic mechanism of L-asparagine and glycoasparagines will be discussed.
Subject(s)
Asparagine/metabolism , Aspartylglucosylaminase/metabolism , Recombinant Proteins/metabolism , Ammonia/metabolism , Asparagine/pharmacology , Aspartic Acid/metabolism , Binding Sites , Binding, Competitive , Catalysis , Chromatography, High Pressure Liquid , Humans , Hydrogen-Ion Concentration , HydrolysisABSTRACT
Glycosylasparaginase (EC 3.5.1.26) is an amidase, which cleaves the N-glycosidic linkage during glycoprotein degradation leading to the liberation of L-aspartic acid from various glycoasparagines. In this work we demonstrate that glycosylasparaginase is also capable of catalyzing the synthesis of the N-glycosidic bond by N-beta-aspartylation of beta-glycosylamine using 1-amino-N-acetylglucosamine as the nucleophile and L-aspartic acid beta-methyl ester as the beta-aspartyl donor. Kinetic studies indicated that beta-glycosylamine has 1390-fold higher reactivity than water in the de-beta-aspartylation of the beta-aspartylenzyme, indicative of the presence of a beta-glycosylamine binding sub-site at the substrate binding site of glycosylasparaginase. The reaction can be applied to glycosylaparaginase-catalyzed biosynthesis of novel glycoasparagines.
Subject(s)
Aspartic Acid/chemistry , Aspartylglucosylaminase/chemistry , Glucosamine/chemistry , Humans , Leukocytes/enzymologyABSTRACT
P alpha-Methyl thymidine triphosphate was prepared through the pyrophosphorolysis of P alpha-methyl thymidine diphosphate P beta-diphenyl ester and tested as an alternative substrate for E. coli DNA polymerase 1 (Klenow fragment) using several template-primer systems requiring the formation of 1 to 42 methylphosphono diester bonds. The enzyme catalyzes the incorporation of a P-methyl thymidylic residue with (Sp)-configuration at a single site in a recessive 3'-end as well as at multiple sites along a growing 167 nucleotide long chain. The synthesis of a full length product, containing 42 sites of methylphosphonate incorporation was observed.
Subject(s)
DNA, Antisense/biosynthesis , DNA, Antisense/chemical synthesis , Base Sequence , DNA Polymerase I/metabolism , DNA Primers/chemistry , DNA, Antisense/chemistry , Escherichia coli/enzymology , Kinetics , Molecular Sequence Data , Thymine Nucleotides/chemical synthesis , Thymine Nucleotides/chemistryABSTRACT
The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1' residue and predilection for bulky hydrophobic P2' residues. A synergism in the action of S1' and S2'subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.
Subject(s)
Glycine/analysis , Peptides/metabolism , Serine Endopeptidases/metabolism , Amino Acid Sequence , Binding Sites , Hydrolysis , Kinetics , Oligopeptides/metabolism , Substrate Specificity , ortho-Aminobenzoates/metabolismABSTRACT
By simple substitution of an N-acyl group for the anthraniloyl(o-aminobenzoyl) group, chromogenic p-nitroanilide substrates are converted into highly sensitive fluorogenic substrates of proteases. The fluorescence of the anthraniloyl group is completely quenched by the p-nitroanilide moiety in the intact substrates and is released during their enzymatic hydrolysis. The approach is exemplified by the synthesis of anthraniloyl-Phe p-nitroanilide, anthraniloyl-Lys p-nitroanilide, and anthraniloyl-Gly-Gly-Phe p-nitroanilide as substrates for chymotrypsin, trypsin, and alkaline mesentericopeptidase, respectively. The kinetic parameters of these substrates are slightly better than those of similar derivatives bearing other acyl groups, suggesting that the enhanced sensitivity is completely due to the method of measurement. Since the conversion does not affect the chromogenic properties of the substrates, the same compounds can be used as usual p-nitroanilide substrates as well.
Subject(s)
Chromogenic Compounds , Peptide Hydrolases/analysis , Peptides , ortho-Aminobenzoates , Amino Acids , Anilides , Hydrolysis , Kinetics , Spectrometry, Fluorescence , Substrate SpecificityABSTRACT
The partitioning of the acylenzyme acetyl-(Gly)n-Phe(NO2)-chymotrypsin (n = 0,1,2) to peptide and peptide acid is observed spectrophotometrically. Values of partitioning ratios for various nucleophiles are calculated from the spectral data. They are a measure for the "true" nucleophile reactivity and are useful in the prediction of the best experimental conditions in enzymic peptide synthesis. A large difference in the nucleophile reactivity is observed which is attributed to the S'2-P'2 interaction.
Subject(s)
Chymotrypsin/metabolism , Peptides/chemical synthesis , Animals , Cattle , Indicators and Reagents , Kinetics , Pancreas/enzymology , Substrate SpecificitySubject(s)
Endopeptidases/metabolism , Peptide Biosynthesis , Acylation , Amines/metabolism , Esters , Hydrolysis , Kinetics , ThermodynamicsABSTRACT
The substituent dependence for kcat/Km of trypsin anilide hydrolysis is consistent with a rate-limiting general acid-base catalysed breakdown of a tetrahedral intermediate. The formation and disappearance of this intermediate during the hydrolysis of alpha-N-acetyl-L-lysin p-nitroanilide is observed in stopped-flow experiments.