Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase:: interactions of the GOasesemi state with different buffers, its redox activity and ability to bind azide

被引:20
作者
Saysell, CG
Barna, T
Borman, CD
Baron, AJ
McPherson, MJ
Sykes, AG
机构
[1] Univ Newcastle Upon Tyne, Dept Chem, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England
[2] Univ Leeds, Dept Biochem & Mol Biol, Leeds LS2 9JT, W Yorkshire, England
来源
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY | 1997年 / 2卷 / 06期
基金
英国工程与自然科学研究理事会;
关键词
galactose oxidase variant; Trp290His GOase; buffer interactions; stability constants; redox properties;
D O I
10.1007/s007750050186
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short similar to 8 Angstrom access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr272, reduction potential 400 mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25 degrees C, I=0.100;M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the nonradical GOase(semi) (CUII) form of the Trp290His variant. Equilibrium constants K-eq/M-1 from absorbance changes at 635 nm are for 1:1 interactions with the OH-containing buffers H2PO4- (231), Hepes (43) and Tris (202), concentrations 0-60 mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at similar to 635 nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOase(ox)/GOase(semi) couple is 730 mV, which compares with 400 mV for the WT GOase couple. Consistent with the 730 mV value the GOase(semi) form is not oxidised with [Fe(CN)(6)](3-) (410 mV) or [W(CN)(8)](3-) (530 mV), and much stronger oxidants such as [Mo(CN)(8)](3-) (800 mV) and [IrCl6](2-) (890 mV) are required. The GOase(ox) product is unstable and decays within 20 min with re-formation of GOase(semi). From changes in UV-Vis spectra with pH, Trp290His GOase(semi) gives a pK(a) of 6.9, and rate constants for the oxidation of GOase(semi) with [Mo(CN)(8)](3-) are dependent on this same pK(a). The latter compares with 7.9 for WT GOase(semi), and is assigned here also as protonation of Tyr-495. The 1:1 binding of azide at the substrate-binding (H2O) site of Trp290His GOase(semi) was studied and gives a formation constant 330 M-1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOase(semi). The trends observed indicate less affinity of Trp290His GOase(semi) for the ionic reactants H+ and N-3(-).
引用
收藏
页码:702 / 709
页数:8
相关论文
共 24 条
  • [1] AVIGAD G, 1962, J BIOL CHEM, V237, P2736
  • [2] BARON AJ, 1994, J BIOL CHEM, V269, P25095
  • [3] BERGMAN SG, 1996, INORG CHEM, V5, P1208
  • [4] LUTIDINE BUFFERS OF VERY LIMITED COORDINATION POWER FOR THE PH RANGE 3-8
    BIPS, U
    ELIAS, H
    HAURODER, M
    KLEINHANS, G
    PFEIFER, S
    WANNOWIUS, KJ
    [J]. INORGANIC CHEMISTRY, 1983, 22 (26) : 3862 - 3865
  • [5] Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen
    Borman, CD
    Saysell, CG
    Sykes, AG
    [J]. JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY, 1997, 2 (04): : 480 - 487
  • [6] Good N E, 1972, Methods Enzymol, V24, P53
  • [7] HAMILTON GA, 1981, COPPER PROTEINS META, V3, P193
  • [8] CRYSTAL-STRUCTURE OF A FREE-RADICAL ENZYME, GALACTOSE-OXIDASE
    ITO, N
    PHILLIPS, SEV
    YADAV, KDS
    KNOWLES, PF
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1994, 238 (05) : 794 - 814
  • [9] NOVEL THIOETHER BOND REVEALED BY A 1.7-A CRYSTAL-STRUCTURE OF GALACTOSE-OXIDASE
    ITO, N
    PHILLIPS, SEV
    STEVENS, C
    OGEL, ZB
    MCPHERSON, MJ
    KEEN, JN
    YADAV, KDS
    KNOWLES, PF
    [J]. NATURE, 1991, 350 (6313) : 87 - 90
  • [10] ITO N, 1995, METHOD ENZYMOL, V258, P235