Authors: Griessler, Richard; Psik, Barbara; Schwarz, Alexandra; Nidetzky, Bernd

Source: FEBS Journal, Volume 271, Number 16, August 2004 , pp. 3319-3329(11)

Publisher: Wiley-Blackwell

Abstract:

Using 0.4 mimidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5′-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 °C was, respectively, four- and threefold reduced in two mutants, Arg234→Ala and Arg242→Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem.270, 2126-2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5′-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234→Ala and Arg242→Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K_app), compared to the wild-type (K_app≈ 6 mm). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but ≈ 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK_a values for the cofactor 5′-phosphate and the exogenous oxyanion.

Keywords: apo-phosphorylase; α-glucan; glycogen; maltodextrin; pyridoxal 5′-phosphate

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1432-1033.2004.04265.x

Publication date: 2004-08-01

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• In this Subject: Anatomy & Physiology ,  Biology ,  Chemistry (General) ,  Biochemistry
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