Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies pptx

The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies Christiane Goedl and Bernd Nidetzky Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria Glycosyltransferases (GTs) constitute a diverse class of enzymes that catalyze the synthesis of glycosidic bonds in oligosaccharides and glycoconjugates. A nucleotide-, phospho- or lipid-phospho-activated sugar is typically utilized as the donor substrate, and transfer of the gly- cosyl moiety to the acceptor molecule occurs with either inversion or retention of configuration at the reactive anomeric carbon [1]. After detailed studies of glycogen phosphorylase spanning many decades [2–6], there has been recent rekindled interest in the mecha- nistic characterization of retaining glycosyltransferases, particularly in relation to glycoside hydrolases, the physiological counterpart enzymes that catalyze the breakdown of glycosidic linkages [7]. The canonical Keywords catalytic mechanism; chemical rescue; family GT-4; glycosyltransferase; a-retaining glucosyl transfer Correspondence B. Nidetzky, Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12 ⁄ I, 8010 Graz, Austria Fax: +43 316 873 8434 Tel: +43 316 873 8400 E-mail: bernd.nidetzky@tugraz.at (Received 5 October 2007, revised 10 December 2007, accepted 19 December 2007) doi:10.1111/j.1742-4658.2007.06254.x Schizophyllum commune a,a-trehalose phosphorylase utilizes a glycosyl- transferase-like catalytic mechanism to convert its disaccharide substrate into a-d-glucose 1-phosphate and a-d-glucose. Recruitment of phosphate by the free enzyme induces a,a-trehalose binding recognition and promotes the catalytic steps. Like the structurally related glycogen phosphorylase and other retaining glycosyltransferases of fold family GT-B, the trehalose phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where X is any amino acid) comprising key residues of its putative phosphate- binding sub-site. Loss of wild-type catalytic efficiency for reaction with phosphate (k cat ⁄ K m =21000m )1 Æs )1 ) was dramatic (‡10 7 -fold) in purified Arg507 fi Ala (R507A) and Lys512 fi Ala (K512A) enzymes, reflecting a corresponding change of comparable magnitude in k cat (Arg507) and K m (Lys512). External amine and guanidine derivatives selectively enhanced the activity of the K512A mutant and the R507A mutant respectively. Analysis of the pH dependence of chemical rescue of the K512A mutant by propargylamine suggested that unprotonated amine in combination with H 2 PO 4 ) , the protonic form of phosphate presumably utilized in enzymatic catalysis, caused restoration of activity. Transition state-like inhibition of the wild-type enzyme A by vanadate in combination with a,a-trehalose (K i = 0.4 lm) was completely disrupted in the R507A mutant but only weakened in the K512A mutant (K i = 300 lm). Phosphate (50 mm) enhan- ced the basal hydrolase activity of the K512A mutant toward a,a-trehalose by 60% but caused its total suppression in wild-type and R507A enzymes. The results portray differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis, reactant state binding of phosphate and selective stabilization of the transition state respectively. Abbreviations G1P, a- D-glucose 1-phosphate; GTs, glycosyltransferases; K512A, Lys512 fi Ala mutant; R507A, Arg507 fi Ala mutant; ScTPase, Schizophyllum commune trehalose phosphorylase; S N i-like, internal return-like mechanism. FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 903 mechanism of a retaining glycoside hydrolase is that of a double-displacement reaction involving a covalent glycosyl-enzyme intermediate [4,8–10]. Because evi- dence from structural and mutagenesis studies has so far failed to support a similar type of intermediate for retaining glycosyltransferases [6,7,11–14], an alternative mechanistic scenario termed internal return-like (S N i- like) was considered in which hypothetically, direct front-side displacement of the leaving group by the incoming nucleophile would result in retention of the anomeric configuration (Fig. 1). Reaction is proposed to occur via a single transition state featuring a highly developed oxocarbenium-ion character. A strict requirement for the tentative S N i-like mechanism is that donor and acceptor substrates are precisely posi- tioned in close proximity to each other in the enzyme active site. Indeed, reaction through a ternary complex where both substrates must bind to the enzyme before the first product is released appears to be a defining catalytic feature of retaining glycosyltransferases; structural insights into enzymatic glycosyl transfer via a ternary complex are provided elsewhere [3,5,12,15]. However, little is known about catalytic factors that could facilitate enzymatic glycosyl transfer via the pro- posed S N i-like process, and the stabilization of the oxocarbenium ion-like species in the transition state. The present study was concerned with the quantitative analysis of the role of noncovalent interactions between active-site residues of the enzyme and the phosphate nucleophile ⁄ leaving group in a reaction cat- alyzed by a sugar 1-phosphate dependant transferase. Schizophyllum commune trehalose phosphorylase (ScTPase; EC 2.4.1.231) utilizes a glycosyltransferase- like catalytic mechanism to convert a,a-trehalose and phosphate into a-d-glucose 1-phosphate (G1P) and a-d-glucose in a freely reversible reaction [16,17]. In the direction of phosphorolysis, recruitment of phos- phate by the free enzyme induces binding recognition for a,a-trehalose and promotes the catalytic steps of glucosyl transfer. d-glucose is released from the ternary enzyme–product complex, and dissociation of G1P regenerates the free enzyme [17]. The unreactive phos- phate-analogue vanadate is a transition state-like inhibitor of ScTPase [18,19] whereby partial mimicry of the transition state was proposed to derive from a hydrogen bond between vanadate and the a-anomeric hydroxyl of the glucose leaving group ⁄ nucleophile (Fig. 1). Unlike glycogen phosphorylase, which utilizes pyridoxal 5¢-phosphate to promote the attack of the phosphate [5] and other nucleotide sugar-dependent glycosyltransferases, which often require a metal ion for activation of the leaving group [20–22], ScTPase does not employ a cofactor in catalysis. Based on sequence similarity, ScTPase has been clas- sified into family (GT)-4 of the glycosyltransferase fam- ilies. Recent crystal structures of three representatives of family GT-4 [23,24] revealed a common protein structural organization typical of transferases of fold family GT-B where the catalytic centre, which features a highly conserved architecture, is situated in a deep cleft formed by two Rossman-fold domains. The struc- ture of Mycobacterium smegmatis phosphatidylinositol- mannosyltransferase (PimA) in complex with the natural sugar-donor substrate GDP-mannose showed that the distal phosphate moiety of the GDP leaving group was tightly coordinated by strong hydrogen bonds with Gly16, Arg196 and Lys202 [23]. The suggestions from a mutational analysis of ScTPase that the homologous Gly292, Arg507 and Lys512 (see supplementary Fig. S1) serve a key role in the phosphorylase reaction as phosphate-binding residues are thus strongly sup- ported [16]. Numbering of ScTPase starts with the initiator methionine as 1 and does not consider the N-terminal 11 amino acid-long fusion peptide that is used for recombinant protein production. In the pres- ent study, we have extended significantly the previous Fig. 1. Reaction of trehalose phosphorylase via an S N i-like mechanism proposed for retaining glycosyltransferases where direct front-side displacement results in retention of anomeric configuration. A predicted gen- eral feature of the mechanism is the devel- opment of a strong hydrogen bond between the incoming nucleophile and the leaving group in the transition state of the reaction. The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky 904 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS scanning mutagenesis of the active site of trehalose phosphorylase [16] and report on results of steady- state kinetic analysis and chemical rescue studies for site-directed ScTPase mutants Arg507 fi Ala (R507A) and Lys512 fi Ala (K512A). A detailed portrait of the catalytic function of the two basic side chains is pro- vided. These new insights are of general interest con- sidering the presence of an active-site consensus motif, Arg507-XXXX-Lys512 (where X is any amino acid) as in ScTPase, in glycogen phosphorylase [3,6] and other retaining clan IV glycosyltransferases of fold family GT-B [12,23,25]. Interestingly, mammalian and yeast glycogen synthases of family GT-3 appear to have replaced the Arg-XXXX-Lys motif of their bacterial and plant counterpart enzymes in family GT-5 by two conserved clusters of Arg residues [26,27]. Likewise, GT-A fold glycosyltransferases also utilize a conserved diad of Arg and Lys for binding of the donor substrate pyrophosphate group and in catalysis. The two basic residues occur in a motif displaying the inverted GT-B pattern, Lys359-XXXXX-Arg365 as in bovine a-1,3- galactosyltransferase of family GT-6 [28]. Despite the evidence provided by the high-resolution crystal struc- tures for many of these glycosyltransferases, the role of the conserved residues for promoting a-retaining glycosyl transfer has not been well defined using muta- genesis and detailed kinetic analysis. Results Analysis of kinetic consequences in R507A and K512A mutants of ScTPase CD spectra of purified wild-type and mutant trehalose phosphorylases were almost superimposable on each other (see supplementary Fig. S2), indicating that the relative proportion of secondary structural elements in the folded structure of the wild-type enzyme was not altered significantly in R507A and K512A mutants. Therefore, this strongly suggests that kinetic conse- quences resulting from the replacement Arg507 fi Ala or Lys512 fi Ala are not due to partial misfolding of the mutant enzymes. Figure 2 displays results of the steady-state kinetic characterization of R507A and K512A, and kinetic parameters of wild-type and mutant phosphorylases for phosphorolysis of a,a-tre- halose are summarized in Table 1. Both R507A and K512A exhibited a dramatic ( ‡10 7 -fold) loss of cata- lytic efficiency for reaction with phosphate (k cat ⁄ K m ) in comparison with the wild-type enzyme. In R507A, the effect on k cat ⁄ K m was distributed between a major, 4.1 · 10 5 -fold decrease in apparent catalytic centre activity (k cat ) and a comparably minor, 130-fold increase in the value of K m for phosphate. In K512A, by contrast, the initial phosphorolysis rate was linearly dependent on the concentration of phosphate up to 1.5 m (Fig. 2), precluding determination of k cat and K m Fig. 2. Steady-state kinetic characterization of R507A (s) and K512A (d) mutant trehalose phosphorylases. Reaction rates (V) were recorded in 50 m M Mes buffer, pH 6.6, using a constant con- centration of 400 m M a,a-trehalose. [E] is the molar enzyme con- centration. Reaction mixtures were incubated at 30 °C for up to 40 h, and the release of G1P was determined enzymatically. A plot of concentration of G1P released against the incubation time was linear in all cases, allowing determination of V and showing that both enzymes were reasonably stable under the incubation condi- tions. Error bars indicate the SD of four independent determina- tions. Table 1. Comparison of kinetic parameters for wild-type, R507A and K512A mutant trehalose phosphorylases in a,a-trehalose phosphoroly- sis direction at 30 °C and pH 6.6. k cat ⁄ K m phosphate [10 )3 ÆM )1 Æs )1 ] Fold decrease K m phosphate [mM] Fold increase K ic vanadate [lM] Fold increase V hydrolysis ⁄ [E] [10 )4 Æs )1 ] 0m M phosphate a 50 mM phosphate ScTPase 2.1 · 10 7 ± 0.4 · 10 7 1 0.8 ± 0.1 1 0.4 ± 0.1 1 3.5 ± 0.4 No hydrolysis R507A 0.4 ± 0.1 5.3 · 10 7 103 ± 32 129 No inhibition 5.0 ± 0.6 No hydrolysis K512A 1.4 ± 0.2 1.5 · 10 7 No saturation 298 ± 51 745 15 ± 1.2 24 ± 2.3 a Data from [16]. C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 905 as independent kinetic parameters. The value of k cat ⁄ K m was obtained from the slope of the straight line fitted to the data in Fig. 2. It was 1.5 · 10 7 -fold lower than the corresponding catalytic efficiency of the wild-type enzyme. The decrease in k cat ⁄ K m for K512A must therefore reflect a very large (>2000-fold) increase in K m , compared with the wild-type values, suggesting that the site-directed replacement of Lys512 caused a more substantial disruption of binding affin- ity for phosphate than that of Arg507. Table 1 also summarizes values of K ic vanadate for wild-type and mutant trehalose phosphorylases. As in the wild-type enzyme, vanadate acted as a competitive inhibitor against phosphate in K512A. However, K ic vanadate was increased 745-fold as result of the site- directed replacement Lys512 fi Ala. By contrast, R507A was not at all inhibited by the used concentra- tions of vanadate (0.5–5.0 mm). The ratio of K m ⁄ K ic vanadate was therefore changed as result of the site-directed substitution of Arg507 from a value of 2000 in the wild-type enzyme to an infinitesimally small value (=103 ⁄¥) in the mutant. It appears to have been increased to a value significantly >2000 (=1600 ⁄ 0.3) in K512A. The hydrolase activities of wild-type and mutant tre- halose phosphorylases towards a,a-trehalose were com- pared under hydrolysis-only conditions [16] and under conditions where, in the presence of 50 mm of phos- phate, phosphorolysis competed with hydrolysis of the disaccharide. The results are summarized in Table 1. Inhibition by vanadate of the hydrolysis of a,a-treha- lose and G1P catalyzed by wild-type ScTPase was also measured. With the methods used, it was not possible to quantify, in the wild-type, a small proportion of a,a-trehalose conversion by ‘error hydrolysis’ next to an overwhelmingly predominant phosphorolysis reac- tion, which also produces d-glucose. However, within limits of detection of the experimental procedures (£1%), no hydrolysis of a,a-trehalose by wild-type enzyme took place when 50 mm of phosphate was present. Because replacement of Arg507 caused selec- tive slowing down of the phosphorolysis reaction com- pared with the hydrolysis of a,a-trehalose [16], the complete suppression of the hydrolase activity of R507A towards a,a-trehalose upon addition of 50 mm of phosphate could be established unambiguously. By marked contrast, the basal rate of hydrolysis of a,a-trehalose by K512A was enhanced significantly (approximately 1.6-fold) in the presence of 50 mm of phosphate. By contrast to the clear inhibitory effect of vanadate on the hydrolysis of a,a-trehalose by wild- type ScTPase (3.5-fold), vanadate did not inhibit the hydrolysis of G1P by the same enzyme. Values of V hydrolysis ⁄ [E] were 4.0 · 10 )4 Æs )1 and 3.9 · 10 )4 Æs )1 in the absence and presence of vanadate, respectively. Noncovalent complementation of trehalose phosphorylase activity in R507A and K512A Inclusion of 200 mm of guanidine into the assay for phosphorolysis of a,a-trehalose at pH 6.6 caused 45-fold enhancement of the activity of R507A seen in the absence of guanidine (k 0 = 4.2 · 10 )5 Æs )1 ). Likewise, a 23-fold stimulation of the basal activity of K512A (k 0 = 6.1 · 10 )5 Æs )1 ) was observed in the presence of 200 mm of propargylamine. Functional complementation of R507A and K512A, expressed as k rescue ⁄ k 0 , displayed a hyperbolic dependence on the concentration of the respective rescue reagent (Fig. 3). Values of k max (R507A: 2.8 · 10 )3 Æs )1 ; K512A: 1.6 · 10 )3 Æs )1 ) and K R (guanidine, 100 mm; propargyl- amine, 58 mm) were obtained with a relative SD of approximately 6% and 10%, respectively, using non- linear fits of Eqn (1) to initial-rate data recorded in the absence and presence of rescue reagent concentra- tions in the range 10–200 mm. Guanidine and propar- gylamine did not exhibit a significant effect on the activity of the wild-type enzyme, except for a weak inhibition (<50% reduction in rate) by concentrations of guanidine higher than 100 mm. No cross-reactiva- tion of R507A by propargylamine (10–200 mm) and K512A by guanidine (10–200 mm) was observed. Addi- tion of 200 mm of NaCl did not alter the activity of either one of the site-directed mutants. These results Fig. 3. Functional complementation of mutant trehalose phosphory- lases. R507A was reactivated by guanidine (s) and K512A by prop- argylamine (d). No cross-reaction was observed, and the rescue agents did not significantly alter the wild-type activity (guanidine ,, propargylamine ). Lines show the fit of Eqn (1) to the data. The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky 906 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS provide strong evidence against the possibility of a non- specific activation of ScTPase mutants and suggest that external guanidine and propargylamine can partly com- pensate for the loss of the original side chain in R507A and K512A, respectively. A series of primary amines and derivatives of guani- dine were therefore examined for their ability to restore phosphorylase activity in K512A and R507A, respec- tively. The results obtained are summarized in Table 2, along with relevant structural and electronic parameters of the compounds used for chemical rescue. Because k rescue for R507A and K512A appeared to exhibit a complex dependence on steric factors of the rescue reagents and the set of amine and guanidine compounds tested was rather small (five each), we did not pursue construction of the respective Brønsted plot using quan- titative structure–activity relationship analysis. How- ever, even in the absence of correction for the influence of molecular volume and hydrophobicity of the rescue reagent, Table 2 shows clearly that the effect of the pK a of the amine and guanidine derivatives on specific resto- ration of activity in K512A and R507A, respectively, was very small and probably not significant. Analysis of the pH dependence of functional complementation of K512A k max ⁄ K R for chemical rescue of K512A by propargyl- amine and R507A by guanidine was pH-dependent. Its value decreased in K512A from 0.035 m )1 Æs )1 at pH 6.6 to 0.003 m )1 Æs )1 at pH 8.2, and in R507A from 0.028 m )1 Æs )1 at pH 6.6 to 0.005 m )1 Æs )1 at pH 8.0. These pH effects are explicable on account of changes in the ionization states of the enzyme and the substrate phosphate (pK a,2 = 7.2) and, in the case of K512A, deprotonation of propargylamine at high pH (pK a = 8.2). Analysis of pH-rate profiles for wild-type ScTPase suggested that H 2 PO 4 ) is the protonic form of phosphate utilized in the enzymatic reaction [17]. The pH-dependence of functional com- plementation of K512A was therefore examined in more detail. Figure 4 compares pH-rate profiles of K512A assayed in the absence and presence of 200 mm of propargylamine with the corresponding pH-rate pro- file of the wild-type enzyme (Fig. 4A) and summarizes the results of chemical rescue experiments with K512A carried out at four different pH values (Fig. 4B). Figure 4A shows that pH-rate profiles of wild-type enzyme and K512A were similar, both showing maximum enzyme activity at an approximate pH of 6.5. Addition of propargylamine caused an up- shift of the optimum pH of K512A by approximately 1 pH unit. Restoration of trehalose phosphorylase activity in K512A by propargylamine was best at pH 7.5 where a value of 140 was observed for k rescue ⁄ k 0 when the concentration of rescue reagent was saturating (Fig. 4B). The presence of 100 mm of propargylamine caused an approximately 10-fold enhancement of the catalytic efficiency of K512A for reaction with phosphate at pH 6.6, in reasonable agreement with the results obtained in activity assays at a single phosphate concentration of 50 mm (Table 2). Likewise, under conditions of chemical res- cue of K512A by 200 mm of propargylamine, k cat ⁄ K m for phosphate increased from a value of 0.014 m )1 Æs )1 at pH 6.6 to 0.023 m )1 Æs )1 at pH 7.5, suggesting that the corresponding pH-rate profile in Fig. 4A reflects the pH dependence of k cat ⁄ K m . These results indicate that, for optimum restoration of activity in K512A, the protonation states of propargylamine and phosphate must be matched. We found that there was a good linear correlation between log(k rescue ⁄ k 0 ) and the limiting concentration of either one of the Table 2. Chemical rescue analysis for R507A and K512A. The concentration of external reagent was 200 mM. Values of V ⁄ [E] were recorded at 30 °Cin50m M Mes buffer, pH 6.6, using 400 mM a,a-trehalose and 50 mM potassium phosphate as substrates. Molecular volume (Mol. volume) and hydrophobicity (logP) were calculated using the programs SPARTAN 06, version 1.1.0 and KOWWIN, respectively. pK a values are from the literature [32,39]. R507A pK a Molecular volume [A ˚ 3 ] logP V ⁄ [E] [10 )4 Æs )1 ] a Fold increase a K512A pK a Molecular volume [A ˚ 3 ] logP V ⁄ [E] [10 )4 Æs )1 ] a Fold increase a No additive 0.42 1 No additive 0.61 1 Guanidine 13.6 59.1 )1.63 19 45 Methylamine 10.6 46.5 )0.64 10 16 Methylguanidine 13.4 79.5 )1.16 5.2 12 Ethylamine 10.6 65.0 )0.15 24 39 Ethylguanidine 13.3 98.1 )0.67 5.4 13 Ammonia 9.2 25.4 )1.38 0.9 1.5 Acetamidine 12.5 67.7 )2.52 2.0 4.8 Propargylamine 8.2 75.8 )0.43 14 23 (24) b Aminoguanidine 11.0 71.3 )1.99 1.6 3.8 2,2,2-Trifluoro ethylamine 5.7 79.5 0.27 6.7 11 (122) b a V ⁄ [E] measured in the absence and presence of rescue reagent are referred to as k 0 and k rescue in text, respectively. Likewise, k rescue ⁄ k 0 in text corresponds to fold increase. b Values in parentheses were corrected for the fraction of protonated amine. C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 907 compounds, H 2 PO 4 ) and unprotonated amine, in this combination of protonic forms (Fig. 4B, inset). Discussion Evidence from high-resolution X-ray structures [3,12,25,29] and site-directed mutagenesis [13,30,31] of glycosyltransferases of fold family GT-B supports an important role for the consensus motif, Arg507- XXXX-Lys512 as in ScTPase, in binding recognition of the phospho leaving group of the glycosyl donor substrate. However, little is known about the individ- ual contribution of each side chain to catalytic efficiency. The present study of Sc TPase used site- directed replacement by Ala and detailed kinetic comparison of wild-type and mutant enzymes to portray the tasks fulfilled by Arg507 and Lys512 in the interaction network of active site residues during binding of phosphate and in catalysis. Although we consider a-retaining glucosyl transfer via the S N i-like mechanism plausible for ScTPase (Fig. 1), the results presented here do not provide evidence that would settle the mechanistic debate surrounding this and other retaining glycosyltransferases. Proposed roles for Arg507 and Lys512 in the mechanism of ScTPase deduced from analysis of kinetic consequences of their individual replacements by Ala The steady-state ordered kinetic mechanism of ScTPase where phosphate binds before a,a-trehalose [17] implies that k cat ⁄ K m for phosphate is a second-order rate con- stant for the association between the free phosphory- lase and the nucleophile of the reaction. k cat is thought to measure the rate-determining conversion of the ter- nary enzyme–substrate complex [17]. Individual replacements of Arg507 and Lys512 caused disruption of the phosphate binding rate by more than seven orders of magnitude, which is equivalent to an ener- getic destabilization of ‡41 kJÆmol )1 (=RT · ln10 7 where R is the gas constant and T is a temperature of 303.15 °K), compared with the wild-type enzyme. Equi- librium binding in terms of K m for phosphate appeared to be completely destroyed in K512A whereas it was weakened in a comparatively moderate way (130-fold) in R507A. Interestingly, relevant single-site mutants of family GT-35 maltodextrin phosphorylase (Arg535 fi Gln; Lys540 fi Arg) [31] and family GT-5 glycogen synthase (Arg300 fi Ala; Lys305 fi Ala) [13], both from Escherichia coli , showed closely similar K m values to their wild-type forms. Their catalytic cen- tre activities, however, were between three to four orders of magnitude below the corresponding wild-type levels. In k cat terms, the dimension of loss of catalytic activity was significantly higher in R507A than the comparable Arg mutants of the two other transferases. Noteworthy, a Lys211 fi Ala mutant of Acetobacter xylinum a-mannosyltransferase, which shares with ScTPase the membership to family GT-4, was reported to be devoid of any enzyme activity [30]. The crystallo- graphically determined hydrogen bond distance between oxygens of the distal phospho group of UDP- glucose and the side chains of Arg196 and Lys202 of family GT-4 mannosyltransferase PimA was only 2.44 and 2.77 A ˚ , respectively [23]. Removal of either one of the two strong bonds by mutagenesis would therefore be expected to result in marked loss of the binding energy used by the wild-type enzyme to promote the reaction. Kinetic consequences for R507A and K512A mutants of ScTPase are consistent with this structure- derived suggestion. Fig. 4. pH-dependence of functional complementation of K512A by propargylamine. Reaction rates were recorded using 50 mM potassium phosphate and 400 m M a,a-trehalose. (A) pH profiles for wild-type enzyme (d), K512A (s) and K512A determined in the presence of 200 m M propargylamine (.). (B) Chemical rescue of K512A at pH 6.6 (d), 7.5 (s), 8.2 (.) and 8.5 (n). The inset displays the logarithmic dependence of k rescue ⁄ k 0 on the relative content of active compound (H 2 PO 4 ) and NH 2 ) present. The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky 908 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS The comparison of apparent affinities for reversible binding of phosphate (K m ) and the transition state mimic vanadate (K i ) by wild-type and mutant forms of trehalose phosphorylase (Table 1) delineates differen- tial roles for Lys512 and Arg507 in the enzymatic mechanism. The particular change in the ratio of K m ⁄ K i resulting from site-directed substitution of Lys512 (increase) and Arg507 (decrease) compared with the wild-type value suggests that, whereas Lys512 appears to be primarily required for phosphate binding in the reactant state, Arg507 promotes the catalytic step of glucosyl transfer through a selective stabiliza- tion of the transition state of the reaction. Occupancy of the phosphate binding site in wild-type and R507A phosphorylases caused complete shut-down of their hydrolytic activity towards a,a-trehalose in the absence of phosphate whereas, in K512A, addition of phos- phate stimulated weakly the breakdown of the disac- charide via hydrolysis. Steps involved in phosphate binding by the wild-type enzyme arguably include an obligatory exclusion of water from the catalytic site. Their drastic impairment resulting from the site-direc- ted substitution of Lys512 is likely to be responsible for this unusual property of the K512A mutant. Interpretation of results of functional complementation studies Chemical rescue experiments, in which a small mole- cule compensates for the missing side chain of a rele- vant site-directed mutant, often provide valuable insights into the role of active-site arginine [32–36] and lysine residues [37–40] for the catalytic function of dif- ferent enzymes. In the present study, we show that activity lost in Arg507 fi Ala and Lys512 fi Ala vari- ants of ScTPase could be selectively restored by deriv- atives of guanidine and primary amines, respectively. The failure of amines to rescue R507A and, likewise, guanidine derivatives to rescue K512A is consistent with observations made with relevant mutants of sev- eral other enzymes [32–36], and it also supports the notion that Arg507 and Lys512 fulfill different tasks in trehalose phosphorylase catalysis (see above). Partial functional complementation of the catalytic defect in R507A by guanidine displayed saturation behavior with respect to both the rescue agent and the substrate. Therefore, this suggests that guanidine binds to the cleft vacated by the replacement of the side chain of Arg507 in the mutant and both the rescue agent and the substrate phosphate form a ter- nary complex prior to catalysis. Considering a pK a for guanidine of approximately 13.6, we conclude from analysis of the pH dependence of the second- order rate constant for the chemical rescue process that the protonated guanidinium ion is most likely required for noncovalent restoration of phosphory- lase activity in R507A. The observed 5.6-fold dec- rease in k max ⁄ K R in response to an increase in pH from 6.6 to 8.0 would be readily explained by depro- tonation of the phosphate monoanion, which is the form of the substrate presumably utilized in the enzymatic reaction [17], and parallels the effect of the same pH change on k cat ⁄ K m for phosphate in wild-type ScTPase. Chemical rescue of K512A by propargylamine exhibited a complex pH dependence, likely explicable on account of the similar pK a values for the external reagent (pK a = 8.2) and the substrate phosphate (pK a,2 = 7.2). However, the observed pH effects on k max ⁄ K R for propargylamine and k cat ⁄ K m for phos- phate determined in the absence and presence of a sat- urating concentration of propargylamine (200 mm; 4 · K R at pH 6.6) would be best explained if unproto- nated propargylamine and H 2 PO 4 ) were involved in the catalytic reaction of an optimally rescued K512A mutant. The scenario proposed for propargylamine need not be the same for methylamine and ethylamine, which, in spite of their high pK a of 10.6, exhibit comparable efficiency to propargylamine as a rescue reagent of K512A at pH 6.6 (Table 2). Binding of propargylamine to K512A failed to restore, in terms of the K m value, some of the affinity of wild-type treha- lose phosphorylase for phosphate. Therefore, to what extent the function of the original side chain of Lys512 can be gauged by the results of our chemical rescue studies remains elusive. We plotted log(k rescue ⁄ k 0 ) of R507A and K512A against the pK a of the rescue agent taking data from Table 2, assuming that all of the listed derivatives of guanidine and primary amines fit the respective cavity resulting from the replacement of the side chain of Arg507 (98 A ˚ 3 ) and Lys512 (80 A ˚ 3 ) by the side chain of Ala (19 A ˚ 3 ). These limited Brønsted plots did not detect a significant correlation between rescue efficacy and reagent pK a and therefore do not support a role for Arg507 and Lys512 in catalytic proton transfer by ScTPase. However, in the phosphorolysis direction of the enzymatic reaction (Fig. 1), partial protonation of the glycosidic oxygen of a,a-trehalose will be needed to facilitate the departure of the leaving group. In the hypothetical S N i-like catalytic mechanism of the treha- lose phosphorylase, enzyme-bound H 2 PO 4 ) is a strong candidate to fulfill the role of the proton donor. The side chains of Arg507 and Lys512 could provide assis- tance in this process via electrostatic stabilization and positioning of the phosphate ligand. C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 909 Because of the similarity in the reactions catalyzed, it is interesting to compare trehalose phosphorylase with a-1,4-glucan phosphorylase. Crystal structures of E. coli maltodextrin phosphorylase bound with phos- phate and oligosaccharide show that hydrogen bonds from the NE and NH2 atoms of the guanidine side chain of Arg535 to two oxygen atoms of phosphate dominate the contacts between the enzyme and the phosphate group [3,6]. The side chain of Lys540 and the main chain N of Gly115 (corresponding to Gly292 in ScTPase) also interact with the phosphate ligand, which is brought into a plausible catalytic position within hydrogen-bonding distance of the reactive gly- cosidic oxygen of the oligosaccharide. Assuming that a similar network of protein contacts positions vanadate at the active site of ScTPase, mutation of the key Arg507 into Ala would be expected to disrupt the pro- posed hydrogen bond between vanadate and the glyco- sidic oxygen of a,a-trehalose, consistent with the observed complete loss of transition state-like inhibi- tion by vanadate in R507A. In maltodextrin phosphor- ylase, the side chain of Lys540 also hydrogen bonds with the 5¢-phosphate moiety of the pyridoxal phosphate cofactor. These contacts stabilize the cata- lytic 5¢-phosphate group in a position within hydrogen bonding distance of the substrate phosphate from which the attack of inorganic phosphate on the glyco- sidic oxygen is promoted. Furthermore, the highly con- served Glu638 (Glu606 in ScTPase), which is also located at the sugar–phosphate contact region, forms a salt bridge with Lys540. Results from 31 P-NMR stud- ies revealed an indirect interaction of Glu638 with the 5¢-phosphate group of the cofactor [41] and suggested participation of the glutamate in establishing a catalyt- ically relevant network of charged groups in the active site [42]. However, the absence of pyridoxal phosphate in ScTPase implies that the role of the conserved lysine in promoting the enzymatic reaction need not be identical for the two phosphorylases. In summary, based on the evidence obtained in the present study, we propose differential roles for the side chains of Lys512 and Arg507 in trehalose phosphorylase catalysis. Although Lys512 is required for binding of the phosphate nucleophile in the reac- tant state, Arg507 facilitates the reaction through a selective stabilization of the transition state. In the proposed S N i-like mechanism of ScTPase (Fig. 1), electrostatic ‘front-side’ stabilization of the oxocarbe- nium ion-like transition state by the incoming phosphate nucleophile could be a decisive catalytic factor. Arg507 might contribute indirectly to this sta- bilization by bringing the phosphate into a suitable position. Experimental procedures Materials and enzymes Unless otherwise noted, all materials used have been described elsewhere [17,43]. Purified preparations of wild- type ScTPase as well as R507A and K512A mutants thereof were obtained using previously reported procedures [16]. Enzyme stock solutions containing approximately 5 mg proteinÆmL )1 were stored in 50 mm potassium–phos- phate buffer, pH 7.0, and kept at )21 ° C until use. Protein characterization Thawed protein samples were checked by SDS ⁄ PAGE to ensure that partial N-terminal truncation of the phosphor- ylase preparations [16] had not occurred during storage. Far-UV CD spectra of wild-type and mutant phosphory- lases were acquired at 30 °C with a J-715 spectropolari- meter (Jasco Inc., Easton, MD, USA) using a 0.1-cm path length cylindrical cell and instrument settings: step resolution = 0.2 nm; scan speed = 50 nmÆmin )1 ; response time = 1 s; bandwidth = 1 nm. Triplicate spectra were recorded in the wavelength range 260–190 nm using enzymes (approximately 1.6 mgÆmL )1 ) dissolved in 50 mm potassium–phosphate buffer, pH 7.0. They were subse- quently averaged and corrected by a blank spectrum lack- ing enzyme. Smoothing and normalizing was performed using a molecular mass of 82.8 kDa for full-length ScTPase. Protein concentration was determined using the Bio-Rad dye-binding method (Bio-Rad, Vienna, Austria) referenced against BSA as the standard. We are unaware of a method for the titration of active sites in prepara- tions of trehalose phosphorylase. Therefore, the molar enzyme concentration [E] was calculated, in a commonly used procedure, from the concentration of purified pro- tein. Because all enzyme preparations were obtained and treated in exactly the same way and displayed similar sta- bilities of their activities during storage (data not shown), values of [E] for wild-type and mutant enzymes are without internal bias. Steady-state kinetic characterization Buffer exchange to 50 mm Mes, pH 6.6–7.5, and 50 mm Tes, pH 7.5–8.5, was achieved through repeated ultrafiltration of protein stock solutions using 10-kDa cut-off Vivaspin 500 microconcentrator tubes (Sartorius, Gottingen, Germany). Initial rates of phosphorolysis of a,a-trehalose were recorded using a reported discontinu- ous assay [17,43] where the formation of G1P was mea- sured. The concentration of G1P was determined as NADH produced in a second coupled enzymatic reaction catalyzed by phosphoglucomutase and glucose 6-phos- phate dehydrogenase. The reaction mixtures for phospho- The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky 910 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS rolysis had a total volume of 200 lL and were incubated in 1.5-mL tubes at 30 °C, using an Eppendorf Thermo- mixer (Vienna, Austria) for temperature control and gen- tle agitation using instrument settings of 300 r.p.m. Typical enzyme concentrations used were 40 lgÆmL )1 of wild-type and 500 lgÆmL )1 of R507A and K512A. The reaction times varied between 0.1 h for wild-type enzyme and up to 40 h for mutant phosphorylases. A plot of concentration of G1P released against the incubation time was linear in all cases, indicating that enzyme inactivation did not interfere with determination of the initial rate under the conditions used. Enzymatic rates (V) were measured for conditions in which the concentration of phosphate was varied in the range 5–300 mm whereas the concentration of a,a-trehalose was 400 mm and constant. Vanadate added in concentrations of 0.5, 2.5, or 5.0 mm was tested as reversible inhibitor of phosphorolysis of a,a-trehalose catalyzed by wild-type and mutant phos- phorylases at pH 6.6. Inhibition constants (K ic vanadate ) were calculated using initial-rate data acquired under conditions in which the concentration of phosphate was varied at a constant concentration of a,a-trehalose (400 mm) in the absence or presence of different constant concentrations of vanadate. Enzymatic rates of hydrolysis of a,a-trehalose or G1P (V hydrolysis ) were determined at 30 °Cin50mm Mes buffer, pH 6.6, using 400 mm of disaccharide or 50 mm of sugar 1-phosphate substrate and measuring the concentration of d-glucose released in samples taken at different times, up to 48 h. A hexokinase-based spectrophotometric assay was used for the determination of d-glucose. Hydrolytic reac- tions for wild-type phosphorylase were performed in the absence and presence of 20 lm of vanadate. Functional complementation studies for R507A and K512A mutants Initial rate assays in the direction of phosphorolysis of a,a-trehalose were used to analyze restoration of activity in K512A or R507A caused by the addition of an external primary amine or a derivative of guanidine. Experiments were carried out at 30 °Cin50mm Mes buffer, pH 6.6, containing 50 mm of potassium–phosphate and 400 mm of a,a-trehalose. The concentration of the amine or guanidine derivative was typically 200 mM and constant, with the exception of chemical rescue of R507A by guanidine and K512A by propargylamine, which was analyzed at different concentrations of external reagent in the range 10–200 mm. Suitable controls showed that none of the added amines or guanidines had a significant effect on the activity of the wild-type enzyme incubated under otherwise exactly identi- cal conditions to the wild-type enzyme alone. The increase in ionic strength resulting from the addition of amine or guanidine derivative was not corrected. However, the com- parison of initial rates measured in the absence and presence of NaCl in concentrations in the range 10–200 m m at pH 6.6 and 7.5 clearly indicated that the activities of R507A and K512A were not influenced by the relevant ionic strength changes. The ratio k rescue ⁄ k 0 , where k 0 and k rescue are V ⁄ [E] values determined in the absence and presence of chemical rescue agent, respectively, is used to express the degree of activation of the mutant. The pH dependence of functional complementation of K512A by propargylamine was determined in the pH range 6.6–8.5 using different reagent concentrations in the range 10–200 mm. Data processing Processing of initial-rate data for the calculation of kinetic parameters and inhibitor binding constants used reported procedures [17]. Equation (1) was fitted to data from activ- ity restoration experiments where k max is the maximum initial rate, divided by [E], obtained at a saturating concen- tration of the rescue agent, and K R is the half-saturation constant for the reagent. k rescue ¼ k max Á½rescue agent=ðK R þ½rescue agentÞ þ k 0 ð1Þ Acknowledgements Financial support from the FWF Austrian Science Fund (project DK Molecular Enzymology W901-B05) is gratefully acknowledged. We thank Professor Walter Keller (Department of Chemistry, University of Graz) for help with CD spectroscopic analysis. References 1 Coutinho PM, Deleury E, Davies GJ & Henrissat B (2003) An evolving hierarchical family classification for glycosyltransferases. J Mol Biol 328, 307–317. 2 Madsen NB & Withers SG (1986) Glycogen phosphory- lase. 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Characterization of trehalose phosphorylase from Schizophyllum commune Biochem J 341, 385–393 Supplementary material The following supplementary material is available online: Fig S1 Conservation of active -site residues among retaining glycosyltransferases of fold family GT-B displayed in a partial multiple sequence alignment Fig S2 Comparison of CD spectra of wild-type (—), R507A (- - -) and K512A (ÆÆÆÆÆ) mutant trehalose. .. trehalose phosphorylases This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 903–913 ª 2008 The. .. 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The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site- directed mutagenesis and chemical rescue studies Christiane Goedl and Bernd Nidetzky Institute of Biotechnology. feature of the mechanism is the devel- opment of a strong hydrogen bond between the incoming nucleophile and the leaving group in the transition state of the reaction. The phosphate site of Schizophyllum. cross-reactiva- tion of R507A by propargylamine (10–200 mm) and K512A by guanidine (10–200 mm) was observed. Addi- tion of 200 mm of NaCl did not alter the activity of either one of the site- directed mutants. These