ISSN:
1432-1327
Keywords:
Key words Galactose oxidase variant
;
Trp290His GOase
;
Buffer interactions
;
Stability constants
;
Redox properties
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Abstract 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 ∼8 Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400 mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25 °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 non-radical GOasesemi (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 ∼635 nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOaseox/GOasesemi couple is 730 mV, which compares with 400 mV for the WT GOase couple. Consistent with the 730 mV value the GOasesemi 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 GOaseox product is unstable and decays within 20 min with re-formation of GOasesemi. From changes in UV-Vis spectra with pH, Trp290His GOasesemi gives a pK a of 6.9, and rate constants for the oxidation of GOasesemi with [Mo(CN)8]3– are dependent on this same pK a. The latter compares with 7.9 for WT GOasesemi, and is assigned here also as protonation of Tyr-495. The 1 : 1 binding of azide at the substrate-binding (H2O) site of Trp290His GOasesemi 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 GOasesemi. The trends observed indicate less affinity of Trp290His GOasesemi for the ionic reactants H+ and N3 –.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s007750050186
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