Tài liệu Báo cáo khoa học: Relationships between structure, function and stability for pyridoxal

Relationships between structure, function and stability for pyridoxal

5¢-phosphate-dependent starch phosphorylase from Corynebacterium

callunae as revealed by reversible cofactor dissociation studies

Richard Griessler, Barbara Psik, Alexandra Schwarz and Bernd Nidetzky

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria

Using 0.4 M imidazole 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,

Arg234fiAla and Arg242fiAla, 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-phos￾phorylases 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 phos￾phate concentration and results from interactions with the

oxyanion that are specific to the quarternary state.

Arg234fiAla and Arg242fiAla mutants showed, respect￾ively, eight- and > 20-fold decreased apparent affinities for

phosphate (Kapp), compared to the wild-type (Kapp
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 dif￾ferent 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 dif￾ferent for the native enzyme and pyridoxal-phosphorylase

bound to phosphate and could reflect the differences in

pKa values for the cofactor 5¢-phosphate and the exogenous

oxyanion.

Keywords: apo-phosphorylase; a-glucan; glycogen; malto￾dextrin; pyridoxal 5¢-phosphate.

Structure–function relationship studies of a-glucan phos￾phorylases (GP) have a rich history in biochemical litera￾ture. It is well established that pyridoxal 5¢-phosphate (PLP)

is the essential cofactor in all known GPs [1]. PLP is bound

via a Schiff base between its aldehyde group and a

conserved lysine side chain in the active site [1,2]. The

5¢-phosphate group is a main catalytic component of PLP

and is required for GP activity [2]. The functional oligomeric

state of GP is dimeric [3–5]. It has been shown that

dissociation of the subunits under localized denaturing

conditions exposes PLP to solvent. PLP is released from the

enzyme and the activity is lost [6–8]. Apo-phosphorylase can

be reconstituted, either with PLP or a range of structural

analogues thereof [2,9,10]. Whereas restoration of enzyme

activity upon the apofiholo conversion is determined by

cofactor structure, the process of dimerization is relatively

indiscriminate in respect to structural modifications of PLP.

Induction of structural complementarity of the interacting

subunits such that they are able to recognize each other and

associate to dimers is correlated with enzyme–cofactor bond

formation [5,9]. In a thorough investigation, Helmreich and

colleagues prepared a series of hybrid phosphorylases in

which one subunit contained PLP while the other was

bound to an inactive cofactor analogue [5]. They concluded

that intersubunit contacts were also needed to elicit activity

in a potentially active holo-monomer.

With very few exceptions [11,12], the results just sum￾marized were obtained with a single enzyme, GP from

rabbit muscle (RmGP). The activity of RmGP is under the

control of allosteric and covalent regulatory mechanisms

which are different or completely lacking in a large group of

GPs from plants and microorganisms. We therefore asked

the question, what novel information might be gained by

applying the same type of reconstitution experiments

described for RmGP to another phosphorylase from a

different source with different regulatory properties? While

active-site residues are almost invariant in members of the

GP family, the dimer interfaces have been quite variable

during the evolution in respect to the specific interproto￾meric contacts, as revealed by comparative 3D structural

Correspondence to B. Nidetzky, Institute of Biotechnology and Bio￾chemical Engineering, Graz University of Technology, Petersgasse 12/

I, A-8010 Graz, Austria. Fax: +43 316 873 8434,

Tel.: +43 316 873 8400, E-mail: [email protected]

Abbreviations: GP, glycogen phosphorylase; EcMalP, Escherichia coli

maltodextrin phosphorylase; CcStP, Corynebacterium callunae starch

phosphorylase; PLP, pyridoxal 5¢-phosphate; PL, pyridoxal;

RmGP, rabbit muscle GP.

Enzyme: a-glucan phosphorylase or a-1,4-D-glucan:orthophosphate￾a-D-glucosyltransferase (EC 2.4.1.1).

(Received 25 March 2004, revised 21 June 2004,

accepted 22 June 2004)

Eur. J. Biochem. 271, 3319–3329 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04265.x