Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity (original) (raw)

Abstract

The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min.K M values ranging from 30 to 64 µm and from 4 to 36 µm were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-_N_-acetylchitobiose. N-glycosidase A could release even a single_N_-acetylglucosamine, albeit 3000 times slower than a di-_N_-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans α1,3-fucosylated at the asparagine linked_N_-acetylglucosamine irrespective of the presence of xylose in the substrate.

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Abbreviations

CAMCys:

S-carboxamidomethyl cystein

CMCys:

S-carboxymethyl cystein

Fib, Fet, Ova, Taa and Brl:

glycopeptides derived from bovine fibrin, fetuin, ovalbumin, taka-amylase A, and bromelain, respectively

GlcNAc:

_N_-acetylglucosamine

PLA:

phospholipase A2

PNGase:

peptide N-glycosidase

RESOS:

_N_-(Resorufin-4-carbonyl)piperidine-4-carboxylic acid_N′_-hydroxysuccinimide ester

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Authors and Affiliations

  1. Institut für Chemie der Universität fur Bodenkultur Wien, Gregor Mendelstraβe 33, A-1180, Vienna, Austria
    Friedrich Altmann, Stefan Schweiszer & Christoph Weber

Authors

  1. Friedrich Altmann
  2. Stefan Schweiszer
  3. Christoph Weber

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Altmann, F., Schweiszer, S. & Weber, C. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity.Glycoconjugate J 12, 84–93 (1995). https://doi.org/10.1007/BF00731873

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