Elsevier

Carbohydrate Research

Volume 324, Issue 2, 11 February 2000, Pages 107-115
Carbohydrate Research

Hydrolytic activity of α-galactosidases against deoxy derivatives of p-nitrophenyl α-d-galactopyranoside

https://doi.org/10.1016/S0008-6215(99)00281-5Get rights and content

Abstract

The four possible monodeoxy derivatives of p-nitrophenyl (PNP) α-d-galactopyranoside were synthesized, and hydrolytic activities of the α-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger α-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters (Km and Vmax) of the enzymes for the hydrolysis of PNP α-d-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.

Section snippets

1. Introduction

Generally, different types of exo-glycosidases are distinguished on the basis of specificity for the glycon structure of their substrates, and their substrate specificities are expressed in their relative activities against substrates having various aglycons. Indications exist that the specificity of glycosidases for the glycon moiety of the substrate is not absolute. Several studies reported that weak hydrolytic reactions with deoxy analogs of the appropriate glycon were catalyzed by several d

Synthesis of deoxy substrates

The structures of PNP α-d-galactopyranoside (1) and of the newly synthesized deoxy derivatives (7, 16, 23, and 27, Scheme 1) used in the present study are compared in Fig. 1. Each derivative, namely PNP 2-deoxy-α-d-lyxo-hexopyranoside (7), PNP 3-deoxy-α-d-xylo-hexopyranoside (16), PNP 4-deoxy-α-d-xylo-hexopyranoside (23), and PNP α-d-fucopyranoside (27), was obtained in crystalline form. The vicinal diaxial proton coupling constants (J2,3) observed in the 1H NMR spectra of 1, 7, 16, 23, and 27

Materials and methods

New compounds were characterized by elemental analysis and 1H NMR spectra. Melting points were determined with a Yamato model MP-21 capillary apparatus and are uncorrected. Optical rotations were measured with a Perkin–Elmer 141 polarimeter at 20 °C. 1H NMR spectra were recorded with a Varian VXR-400 spectrometer. Chemical shifts are expressed in ppm downfield shift from Me4Si. Mass spectra were obtained with a Jeol JMX SX-102A instrument under positive FAB conditions. Column chromatography was

Acknowledgements

The authors express their sincere thanks to Dr N. Miyata of the National Institute of Health Science for recording the 1H NMR spectra, Dr T. Nakata of the Institute of Physical and Chemical Research (RIKEN) for the elemental analysis, and Ms J. Nakate for the measurement of FAB mass spectra. This work was supported in part by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.

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