Báo cáo khoa học: Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes pot

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Báo cáo khoa học: Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes pot

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Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334 fi Gly mutant enzymes Alexandra Schwarz 1 , Lothar Brecker 2 and Bernd Nidetzky 1 1 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria 2 Institute of Organic Chemistry, University of Vienna, Austria Glycogen phosphorylases are pyridoxal 5¢-phosphate (PLP)-dependent glycosyltransferases (EC 2.4.1.1) that catalyze the reversible phosphorolysis of oligomeric and polymeric a-1,4-glucan substrates (maltodextrins, starch, glycogen) [1,2]. The reaction proceeds with retention of configuration at the anomeric carbon, yielding a-d-glucose 1-phosphate (Glc1P) as product in the direction of substrate depolymerization. In spite Keywords a-retaining glucosyl transfer; phosphorus NMR; pyridoxal 5¢-phosphate; saturation transfer difference NMR; starch phosphorylase Correspondence B. Nidetzky, Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria Fax: +43 316 873 8434 Tel: +43 316 873 8400 E-mail: bernd.nidetzky@tugraz.at (Received 15 May 2007, revised 1 August 2007, accepted 6 August 2007) doi:10.1111/j.1742-4658.2007.06030.x His334 facilitates catalysis by Corynebacterium callunae starch phosphory- lase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disrup- tive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofac- tor pyridoxal 5¢-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (k catP ¼ 0.033 s )1 ) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (K m ¼ 280 mm), respectively. The 31 P chemical shift of pyridoxal 5¢-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334 fi Gly. Analysis of interactions of a-d-glucose 1-phosphate and a-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from satura- tion transfer difference NMR experiments, suggested that disruption of enzyme–substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5–7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced. Abbreviations CcStP, Corynebacterium callunae starch phosphorylase; GL, D-gluconic acid 1,5-lactone; Glc1P, a-D-glucose 1-phosphate; LFER, linear free energy relationship; PLP, pyridoxal 5¢-phosphate; STD, saturation transfer difference; X1P, a- D-xylose 1-phosphate. FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5105 of detailed studies spanning many decades, definite conclusions about the catalytic mechanism of glycogen phosphorylases and the exact function of the PLP co- factor in it are still elusive [2–6]. Figure 1 shows that an active site His has a central role in the contentious debate surrounding a putative covalent glucosyl– enzyme intermediate of a double displacement-like mechanism of the phosphorylase. The precedent of sucrose phosphorylase [7–9], mechanistically represent- ing a large class of retaining glycoside hydrolases and transglycosidases, would strongly favor some form of a two-step mechanism, consisting of glucosylation and deglucosylation of a catalytic group on the enzyme, typically a carboxylate of Glu or Asp [5,10]. Glycogen phosphorylase structures reveal that the backbone amide carbonyl of the His is the only group appropri- ately placed to function as a nucleophile [4,11–15] (Fig. 1B). However, despite the vast assortment of probes used, all searches for a covalent intermediate of glycogen phosphorylase have proved fruitless so far [5]. Partly driven by this negative evidence, an alterna- tive mechanism termed S N i-like was proposed, where, in the direction of phosphorolysis, attack of phosphate as nucleophile and departure of the oligosaccharide leaving group occur on the same face of the glucosyl residue being transferred [2,4–6,11,13]. It involves only a single transition state that has a highly developed oxocarbenium ion character. The His is proposed to stabilize this transition state through electrostatic and hydrogen bonding interactions of its main chain car- bonyl and side chain, respectively. The phosphate ion positioned in a ‘tucked-under’ conformation on the opposite (a) face of the glucosyl oxoarbenium ion-like species presumably provides additional electrostatic stabilization, derived from its interactions with C-1 and O-5 as well as O-2 of the pyranosyl ring [4,6,11,13] (Fig. 1B). Earlier kinetic studies of wild-type phosphorylases support this idea by showing coopera- tive-like (synergistic) binding of phosphate and gluco- syl oxoarbenium ion mimics such as d-gluconic acid 1,5-lactone (GL) [16,17]. Substitution of His334 in starch phosphorylase from Corynebacterium callunae (CcStP) (Fig. 1A) by Gln or Asn caused a substantial (up to 150-fold) loss in wild-type catalytic efficiency that was paralleled by a corresponding decrease in affinity for GL in combination with phosphate, reflect- ing a change from positive to negative cooperativity in binding of the two ligands as a result of the site-direc- ted replacement [18]. In this work, we have substituted His334 with Gly and analyzed the disruptive effects of the point muta- tion on active site function of CcStP using steady-state kinetics and selective NMR probes for the 5¢-phos- phate group of the cofactor and for bound carbo- hydrate ligands. The work was carried out to address three questions in particular, taking into account that, quite unexpectedly, an H334A mutant of CcStP was almost as active as the wild-type enzyme [18]. How does complete removal of the side chain of His334 influence binding and catalysis? Are the properties of neighboring active site groups, including the PLP cofactor, affected by the His fi Gly mutation? If suffi- cient room is vacated in H334G to accommodate water or another nucleophile in place of the original methylimidazole group, will this new ligand participate in the enzymatic reaction such that eventually hydro- N O N N O Pyridoxal P O O OH OH O OH OH OH O P O O O N O N O N O O N N O O O N O O N O N O O His345 (334) Gly114 (114) Leu115 (115) Gly640 (629) Glu637 (626) Tyr538 (527) Asn449 (437) Ser639 (628) 3.0 Å 3.1 Å 2.9 Å 2.9 Å 3.6 Å 3.6 Å 3.6 Å 2.7 Å 3.1 Å 3.7Å 2.7Å 2.8 Å 3.2 Å 4.3 Å 3.2 Å BA Fig. 1. Close-up structure of the active site of CcStP and proposed interactions with Glc1P bound at the catalytic subsite. (A) The picture was generated with PYMOL v.0.99 using X-ray crystallographic coordinates for CcStP with phosphate bound in the active site (Protein Data Bank entry 2C4M). His334, PLP and phosphate are shown as stick models. (B) The scheme was drawn using the structure of E. coli malto- dextrin phosphorylase bound to Glc1P (Protein Data Bank entry 1L5V). Numbering of amino acids is for the E. coli enzyme, and correspond- ing residues of CcStP are given in parentheses. Hydrogen bonds are indicated as broken lines. Role of His334 in a-glucan phosphorylase A. Schwarz et al. 5106 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS lysis or transglucosylation occurs? Mutational analysis of the His homologous to His334 in CcStP has not been performed in another a-glucan phosphorylase. Results Protein purification and cofactor analysis Wild-type CcStP and the H334G mutant were pro- duced in Escherichia coli and purified to apparent homogeneity (data not shown). Both enzymes were obtained in similar yields of about 50%, and contained approximately 0.8 PLPs per subunit of protein. Upon excitation at 330 nm, the wild-type enzyme and the H334G mutant exhibited nearly superimposable cofac- tor fluorescence emission spectra between 470 and 550 nm, with an emission maximum at 520 nm. How- ever, the intensity of cofactor fluorescence at 520 nm in the H334G mutant was only approximately 40% that observed in the wild-type. Characterization of the H334G mutant Enzyme activity The H334G mutant exhibited 0.003% of the wild-type specific activity for phosphorolysis of maltodextrin (33 UÆmg )1 ). External imidazole stimulated activity of the mutant up to 5.5-fold, whereas it weakly inhibited the wild-type (Fig. 2). Acetate and formate had no effect on the activity of the H334G mutant. Azide, 2-methylimidazole and 2-ethylimidazole inhibited the mutant. The wild-type was inhibited weakly (£ 2-fold) by all of the compounds tested, with the exception of formate, which caused a five-fold reduction of activity. Kinetic parameters Steady-state kinetic parameters for phosphorolysis of maltopentaose by the H334G mutant were determined at pH 7.0 under conditions where the concentration of phosphate was constant and saturating (50 mm). The k cat of 0.033 ± 0.001 s )1 was 0.05% of the wild-type value. The K m for maltopentaose was 280 ± 20 mm, reflecting a 75-fold decrease in substrate binding affin- ity as a result of the mutation. Like the wild-type [18], the H334G mutant did not hydrolyze maltopentaose into glucose above a detection limit of about 0.15% of its phosphorylase activity. Ligand binding Dissociation constants (K d ) for complexes of the H334G mutant with GL or Glc1P were obtained from nonlinear fits of a Langmuir binding isotherm to data obtained by fluorescence titration analysis. The K d values were 95 ± 8 lm and 100 ± 10 lm for complexes with Glc1P and GL, respectively. They were decreased seven-fold and three-fold in comparison to K d values for corresponding complexes of the wild-type [18]. The presence of 50 mm phosphate promoted a 30-fold increase in K d (¼ 2.9 ± 0.3 mm) for GL binding to the H334G mutant. This result is in contrast to the 17-fold enhancement of GL binding to the wild-type upon the addition of the same concentration of phosphate. pH profiles The pH dependences of logarithmic rates of the H334G mutant and wild-type are compared in Fig. 3. Data for the wild-type are taken from Griessler et al. [19]. The pH profile of the H334G mutant in the phos- phorolysis direction was a narrow, bell-shaped curve, strikingly different from that of the wild-type and with an optimum pH of 6.5. A shift of the pH profile of about + 0.5–1.0 pH units and an optimum pH similar to that of the wild-type was observed for the H334G mutant in the presence of 200 mm imidazole. By con- trast, the pH rate profile of the wild-type was not affected by addition of the same concentration of imid- azole (data not shown). Phosphorus NMR of pyridoxal 5¢-phosphate 31 P-NMR spectra for solutions of wild-type CcStP and the H334G mutant that contained a similar concentra- tion of enzyme-bound PLP ( 100 lm) were recorded in the pH range 5.6–8.0. Typical spectra acquired at pH 7.25 are shown in Fig. 4A. The 31 P resonance imidazole (mM) 0 rel. activity (-fold) 0 2 4 6 100 200 300 400 500 Fig. 2. Analysis of restoration of activity in wild-type CcStP (d) and the H334G mutant (s) by external imidazole. The results are given as relative specific activities that were normalized by using the spe- cific activities of the wild-type (33 UÆmg )1 ) and the H334G mutant (0.001 UÆmg )1 ) in the absence of imidazole. A. Schwarz et al. Role of His334 in a-glucan phosphorylase FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5107 signal of PLP phosphate in the H334G mutant showed a very low signal-to-noise ratio, necessitating data col- lection for up to 12 h, during which time a perceptible denaturation of the enzyme occurred at pH values below and above 7.25. It was therefore not possible to obtain an exact pH dependence for the 31 P chemical shift of PLP phosphate in the H334G mutant. How- ever, a single 31 P shift at pH 7.25 is provided. Figure 4B compares pH profiles of chemical 31 P shifts for PLP phosphate in wild-type CcStP measured in the absence and presence of 20 mm sodium arsenate. The two pH profiles were almost superimposable on each other. We also determined chemical 31 P shifts at pH 6.68 and 6.93 under conditions in which the pres- ence of arsenate (20 mm) and GL (1 mm) drives formation of a ternary enzyme–ligand complex. The results show that 31 P shifts for PLP phosphate in free enzyme were remarkably insensitive to the binding of arsenate alone and in combination with GL. Analysis of ligand binding by STD NMR Figure 5 summarizes relative saturation transfer differ- ence (STD) effects of Glc1P and a-d-xylose 1-phos- phate (X1P) upon their binding to wild-type and H334G phosphorylase. Glc1P displayed very similar patterns of binding to both enzymes. However, the relative STD effects of the protons in positions 6a and b were slightly higher when Glc1P was bound to the wild-type than when it was bound to the H334G mutant. The relative STD effects of X1P bound to the two enzymes were also fairly similar, with the exception of the proton in position 5eq, which showed a higher effect in the complex with the H334G mutant. Binding of GL to the wild-type and the H334G mutant also yielded very similar STD spectra with, however, quite a low signal-to-noise ratio, very likely caused by the small dissociation constants for enzyme–GL complexes. Appreciable STD effects could be detected only for protons in positions 2 and 4, which caused overlapping signals in the 1 H-NMR spectrum (data not shown) [20]. All other protons showed much lower STD effects, which could not be quantified. Although longer STD measurements could, in principle, improve the signal-to-noise ratio, the duration of the NMR experiment was limited in this case by the spontaneous hydrolysis of GL to gluconic acid. During STD NMR measurements of Glc1P bound to wild-type enzyme, we observed formation of a novel carbohydrate at the expense of Glc1P. This compound was analyzed directly from the NMR sample, and identified as amylose (data not shown). Details underlying the conversion of Glc1P in the pH 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 31 P (p.p.m.) 1 2 3 4 AB Fig. 4. Characterization of PLP phosphate in wild-type CcStP and the H334G mutant using 31 P-NMR. (A) Spectra of wild-type CcStP and the H334G mutant acquired at pH 7.25, and with the number of recorded scans and resulting signal-to-noise ratios indicated. (B) Chemical 31 P shifts of the PLP phosphate resonance signal of wild-type enzyme in the absence of ligand (d), in the presence of 20 m M arsenate (s), and in the presence of 20 m M arsenate and 1 mM GL (.); 31 P shift for the H334G mutant (,), recorded in the absence of arsenate and at only a single pH of 7.25. p H 5.5 log(rel. k cat ) 1.4 1.6 1.8 2.0 6.0 6.5 7.0 7.5 8.0 Fig. 3. pH profiles of catalytic rates for phosphorolysis of maltodex- trin catalyzed by wild-type CcStP (.) and the H334G mutant in the absence (d) and presence (s) of 200 m M imidazole. The initial rates were acquired under conditions of apparent saturation with substrate, and are given as relative values (rel. k cat ) of the catalytic rate for the wild-type (50 s )1 ; pH 7.0) and the catalytic rates of the H334G mutant in the absence (0.0015 s )1 ; pH 6.5) and the pres- ence (0.0081 s )1 ; pH 7.0) of imidazole. The lines indicate the trend of the data. Role of His334 in a-glucan phosphorylase A. Schwarz et al. 5108 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS absence of an exogenous glucosyl acceptor oligosac- charide were not pursued further. Discussion Disruptive effects of active site mutations traced by STD NMR Interpretation of the functional consequences of H334G and active site mutations of enzymes in general is subject to the caveat that site-directed replacement has caused a global change in enzyme–substrate inter- actions occurring in the wild-type. There is a clear need for practical methods capable of characterizing the structural perturbation resulting from site-specific modification of enzyme or substrate with respect to direct as well as indirect disruptive effects caused by it. We would like to suggest the STD NMR technique, which analyzes, in the dissociated ligand, the magneti- zation transferred from protons of the protein to pro- tons of the bound ligand that are in close contact with the protein. Relative STD effects within a given ligand therefore provide a characteristic fingerprint of nonpo- lar ligand interactions within the binding pocket of the protein [21–27]. Because hydrogen bonds and other electrostatic interactions are silent in the STD NMR experiment, the obtained portrait of the binding pat- tern is partial (Fig. 1B), and isolated interpretations of STD effects can therefore be hazardous. However, if STD effects for two minimally modified systems can be investigated and compared, then the interpretation is considerably simplified. The side chain of His334 and the –CH 2 OH group of Glc1P are complementary interacting groups (Fig. 1B), and analysis of changes in relative STD effects resulting from structural pertur- bation of enzyme (H334G) and substrate (X1P) was therefore of particular interest. The results obtained suggest an overwhelmingly local disruption of binding interactions caused by removing the two functional groups individually or together. Analysis of kinetic consequences in the H334G mutant and chemical rescue studies Substitution of His334 with Gly caused a 10 3.5 -fold decrease in the wild-type k cat for phosphorolysis of maltopentaose. Conversion of the ternary enzyme–sub- strate complex is believed to be the rate-determining step of glucosyl transfer to phosphate catalyzed by a-glucan phosphorylases [1], and k catP is the kinetic measure of it. Because substrate binding to enzyme– phosphate is supposed to be a rapid equilibrium pro- cess [1,28], the K m for maltopentaose is an effective dissociation constant that was increased by almost two orders of magnitude in the H334G mutant in relation to the wild-type. Comparison of different CcStP mutants reported here (H334G) and in a recent paper (H334A, H334Q, H334N [18]) reveals that complete removal of the His side chain in the H334G mutant had the largest disruptive effect on both binding and turnover of maltopentaose. Unlike the H334A mutant, in which the kinetic consequences of the site-directed replacement were minimal [18], the H334G mutant had lost  30 kJÆmol )1 of the binding energy used in the wild-type for stabilization of the transition state of the reaction. (The differential binding energy DDG# was Fig. 5. Analysis of sugar 1-phosphate bind- ing to wild-type CcStP and the H334G mutant using STD NMR. Values are relative STD effects of Glc1P bound to wild-type CcStP (Aa) and the H334G mutant (Ab) as well as X1P bound to wild-type CcStP (Ba) and the H334G mutant (Bb). Each STD effect is calculated as a quotient of signal intensities in the STD spectrum and in the reference proton spectrum. The effects are normalized to the respective largest effect in the sample. A. Schwarz et al. Role of His334 in a-glucan phosphorylase FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5109 calculated with the relationship DDG# ¼ RT ln 10 5.2 , using the ratio of k catP ⁄ K m values of 18 000 m )1 Æs )1 and 0.12 m )1 Æs )1 for the wild-type and the H334G mutant, respectively.) We speculated that water might occupy the position vacated in the H334A mutant through removal of the imidazole group of the His, thereby effectively replacing the function of the origi- nal side chain in catalysis by the mutant [18]. What- ever mechanism truly accounts for the retention of phosphorylase activity by the H334A mutant, it is clearly not available to the H334G mutant. The selec- tivity of the H334G mutant for glucosyl transfer to phosphate as compared with water was absolute within the limits of detection of the experimental methods, suggesting that, as in the wild-type and the H334A mutant [18], water was effectively excluded from the reaction with maltopentaose bound to free enzyme or enzyme–phosphate. The notion that substitution of His334 with Gly destabilizes the transition state of glucosyl transfer but otherwise does not alter the course of the reaction cat- alyzed by CcStP is further supported by the results of linear free energy relationship (LFER) analysis and chemical rescue studies. Schwarz et al. [18] have shown that a log–log correlation of catalytic efficiencies of the wild-type and His334 mutants for phosphorolysis of starch with the corresponding reciprocal dissociation constants for GL binding to enzyme–phosphate was linear, with a good coefficient of determination. Using a similar type of correlation, which is now based on k catP ⁄ K m for maltopentaose and includes data for the H334G mutant, we obtain again a plausible LFER with a slope of 1.93 ± 0.45 and a coefficient of deter- mination (r 2 ) of 0.862 (supplementary Fig. S1). A shift in the controlling mechanism of the reaction brought about by the His fi Gly mutation would be expected to cause a breakdown of the LFER, in contrast to the observations made. Whereas external imidazole weakly enhanced the activity of the H334G mutant, it did not participate in the reaction as alternative nucleophile, such that glucose 1-imidazole or the product of its spontaneous hydrolysis (glucose) would be formed in kinetic competition with Glc1P. Other small nucleo- philes, such as azide, were without effect on both activity and reaction course. By way of comparison, when the catalytic nucleophile (Asp) of sucrose phos- phorylase was replaced by Ala, azide could occupy the position of the original carboxylate group and react through addition to C-1 of the glucosyl moiety, yield- ing the inversion product b-glucose 1-azide [9]. We investigated whether the proposed hydrogen bond between His334 and the C-6 hydroxy group of the glucosyl residue bound at the catalytic subsite could become optimized in the transition state. A hypothetical scenario, inspired by studies of human purine nucleoside phosphorylase [29,30], is that His334 could be responsible for positioning O-6 in line with O-5 and the glycosidic oxygen of phosphate (O P1 ) (Fig. 6). In the direction of polysaccharide synthesis, compression of the three-oxygen stack such that O-6 moves closer to the ring oxygen would enhance elec- tron density in the reactive carbon and thus facilitate glycosidic bond cleavage and formation of the transi- tion state in an S N i-like mechanism of glucosyl trans- fer. In the direction of phosphorolysis, both O-6 and the now nucleophilic O P1 of phosphate might be pushed towards O-5 and assist electronically in cataly- sis. As in purine nucleoside phosphorylase [29,30], protein vibrations that are coupled to the reaction coordinate could be responsible for promoting the close approach of the three oxygens. pH rate dependences for the wild-type and the H334G mutant examined with kinetics and 31 P-NMR As for other a-glucan phosphorylases [31–35], the pH profiles of apparent k cat for wild-type CcStP were ND-1 2.7 Å 2.3 Å 3.0 Å 3.8 Å O-6 O-5 O P1 Fig. 6. Suggested role for the hydrogen bond between Nd of His334 and the 6-OH of the glucosyl residue bound at the catalytic subsite in the selective stabilization of the transition state. O-6, the ring oxygen, and the glycosidic oxygen O P1 lie in a close three- oxygen stack that is indicated by a dashed line. Increased electron density near the reactive center provided by squeezing the three oxygens together could facilitate the catalytic step. The picture was generated using Protein Data Bank entry 1L5V (maltodextrin phos- phorylase bound with Glc1P [15]). Role of His334 in a-glucan phosphorylase A. Schwarz et al. 5110 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS bell-shaped curves showing a decrease in activity at low and high pH. Replacement of His334 with Gly caused a marked change in the pH profile of k cat for the phosphorolysis direction. To explore possible sources of the different pH dependences, we used 31 P-NMR and compared chemical shifts for the 5¢-phosphate group of PLP in the wild-type and the H334G mutant. Changes in chemical shift and line width of the 31 P-NMR signal may serve as reporters of alterations in the ionization state of the cofactor phosphate group [36]. They are, however, also expli- cable by changes in the local environment of PLP and their effect on conformational strain on the 5¢-phosphate moiety. The 31 P chemical shift of PLP phosphate in unli- ganded wild-type CcStP was strongly influenced by pH, increasing in a sigmoidal dependence from 1.36 p.p.m. at pH 5.6 to 3.66 p.p.m. at pH 8.0 (Fig. 4B). Slow deprotonation of the triethanolamine buffer interfered with measurement of 31 P chemical shifts in the alkaline region (pH > 7.5), preventing determination of a complete pH profile for the chemical shift and hence calculation of the pK a value of PLP phosphate by curve fitting. However, there is good evidence that the pK a value for PLP phosphate in free Cc StP is ‡ 6.75 (Fig. 4B), and therefore higher than that seen in maltodextrin phosphorylase (pK a ¼ 5.6) [37,38]. To the extent that the shift of the 31 P resonance signal is a sensitive probe of direct contacts between the cofactor 5¢-phosphate group and bound ligands or relevant changes in active site conformation induced by ligand binding [2,38], the evidence for CcStP suggests that the local environment of PLP phosphate remains essentially unaffected upon forma- tion of enzyme complexes with arsenate alone and in combination with GL. By contrast, significant field shifts of the 31 P resonance signal were observed with E. coli maltodextrin phosphorylase [37], potato phos- phorylase [39] and muscle glycogen phosphorylase [40] upon addition of arsenate, probably caused by electrostatic interactions between the 5¢-phosphate moiety and arsenate. The pK a for PLP phosphate in E. coli maltodextrin phosphorylase was also shifted by + 1.1 pH units upon binding of arsenate [37]. Therefore, CcStP appears to differ subtly from mal- todextrin and glycogen phosphorylase in how it copes with constraining the cofactor phosphate group into a configuration that is believed to promote catalysis via direct interaction with the substrate arsenate (or phosphate). A tentative explanation is provided by Fig. 7, which reveals clear differences in the pattern of hydrogen bonding and the orientation of PLP phosphate in the active sites of CcStP bound with phosphate (Fig. 7A) and maltodextrin phosphorylase bound with phosphate and a nonphosphorolyzable substrate analog (omitted in Fig. 7B for reasons of clarity) in Fig. 7B. Gly642 in the E. coli enzyme is substituted by Ser631 in CcStP. Interactions from the main chain amide of Gly are replaced by interactions from both the main chain amide and the side chain of Ser. Hydrogen bonds between PLP phosphate and the side chains of nearby Lys residues and bound phosphate ligand appear to be stronger in maltodex- trin phosphorylase than in CcStP, arguably account- ing for the relative elevation of pK a of PLP phosphate in unliganded CcStP and the apparent lack of perturbation of pK a in the CcStP complex with arsenate. The pH dependence of the 31 P chemical shift of PLP phosphate in wild-type CcStP is not, clearly, borne out in pH rate profiles for the enzymatic reaction. The opti- mum pH range for glucosyl transfer to and from phos- phate overlaps with the pH region (pH 6.0–7.0) where monoanionic and dianionic forms of 5¢-phosphate should both be present in similar relative amounts. The loss of wild-type activity in the direction of synthesis at A B Fig. 7. Comparison of the sites for PLP phosphate in CcStP (A) and E. coli malto- dextrin phosphorylase (B). Pictures were generated using Protein Data Bank entries 2C4M (CcStP) and 1L5W (maltodextrin phosphorylase bound with phosphate and a substrate analog [15]). A. Schwarz et al. Role of His334 in a-glucan phosphorylase FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5111 pH 6.5 [19] may be correlated, at least formally, with the strong field shift of 31 P resonance signal in this pH range, perhaps reflecting the formation of a PLP dian- ion. Electrostatic repulsion may now prevent the cofac- tor 5¢-phosphate and also the dianionic phosphate of the glucosyl donor substrate from closely approaching each other [2,41]. Rather than eliminating a single ionization from pH profiles, substitution of His334 by Gly caused a complex pattern of changes in the pH rate dependenc- es of the wild-type. The acidic and basic limbs on the pH profile of the H334G mutant for the phosphoroly- sis direction were displaced inward by  0.5 pH units in comparison with the corresponding pH profile of the wild-type, and the optimum pH range for the mutant was also shifted, by about ) 0.75 pH units. In addition to partly restoring activity in the H334G mutant, external imidazole caused an upshift by £ 1.0 pH units of the entire pH dependence of phosphoro- lysis by the mutant, whereas the pH rate profile of the wild-type was not influenced by the added imidaz- ole. Although these results suggest that His334 influ- ences the pH dependence of the activity of CcStP, they do not delineate a detailed relationship. Appar- ent ionizations on the pH rate profiles must probably be assigned to pH-dependent ‘titration’ of more than just a single residue. Experimental procedures Materials Materials for mutagenesis, protein purification and enzy- matic assays have been described elsewhere in more detail [18,42]. Restriction endonucleases were obtained from Fer- mentas (St Leon-Rot, Germany). Oligonucleotide synthesis and DNA sequencing was performed at VBC Biotech Ser- vices GmbH (Vienna, Austria). All other chemicals were of the highest quality and were provided by Sigma-Aldrich (Vienna, Austria). Mutagenesis, protein expression and purification The point mutation His334 fi Gly was introduced by the PCR-based overlap extension method [43]. PCR conditions were as described previously [42], except for an annealing and elongation time of 1 min and an annealing temperature of 55 °C. We used the following pairs of flanking and mutagenic primers, where Eco91I and XagI restriction sites are underlined and the mismatched codons are indicated in bold, respectively: XagI-for, 5¢-GGGAACTCTGCG CCT GTGGAAGGC-3¢; Eco91I-inv, 5¢-CTCATCCAGATCG GTTACCCAATC-3¢; H334G-for, 5¢-TACACCAACGGAA CCGTGCTCAC-3¢; H334G-inv, 5¢-GTGAGCACGGTTC CGTTGGTGTACGC-3¢. The plasmid pQE70–CcStP [42] containing the gene for wild-type CcStP was used as the template. The mutagenized plasmid was transformed into E. coli JM109 cells. Protein expression and purification of the H334G mutant were car- ried out using published protocols [18]. Enzyme activity was measured with a continuous coupled assay reported elsewhere [9], and protein was determined by the Bio-Rad (Vienna, Austria) dye binding assay using BSA as standard. Steady-state kinetic analysis and biochemical characterization Initial rates of phosphorolysis were determined in discontin- uous assays as described previously [44]. The enzyme (5.5 lm subunits of the H334G mutant) was incubated at 30 °Cin 300 mm potassium phosphate buffer, and the release of Glc1P was measured as a function of time of incubation up to 3 h. Maltodextrin or maltopentaose was used as the sub- strate, as indicated in Results. The sodium salts of azide, ace- tate, and formate, as well as imidazole, 2-ethylimidazole, and 2-methylimidazole, were tested in the range 10–250 mm for possible restoration of activity of the H334G mutant for phosphorolysis of maltodextrin (23 gÆL )1 ) at pH 7.0. Con- trol reactions with the wild-type were carried out in all cases. The H334G mutant was examined for possible hydrolase activity by incubating the enzyme (6.7 lm)at30°Cin 50 mm triethanolamine buffer (pH 7.0), containing malto- pentaose (75 mm) and potassium sulfate (20 mm). Note that sulfate was added in this series of measurements to ensure stability of the enzymes during the timespan of experiments carried out in the absence of phosphate [45]. Samples were taken at certain time points up to 40 h, and the formation of glucose was measured as described elsewhere [18]. pH dependence studies were performed in the pH range 5.5–8.0. The pH values were adjusted at the temperature of measurement (30 °C), and controlled before and after the enzymatic reaction. Ionic strength changes in the pH range examined were not considered. Catalytic rates of the H334G mutant were acquired under conditions of apparent saturation with substrate (300 mm phosphate, 23 gÆL )1 mal- todextrin). Apparent dissociation constants for the binary complex of the H334G mutant bound with Glc1P or GL, and for the ternary complex of the H334G mutant bound with phosphate and GL, were determined by titration analysis in which quenching of the PLP fluorescence was measured. Following excitation at 330 nm, an emission spectrum in the range 350–550 nm was recorded. The full experimental protocol and details of data processing for the calculation of dissociation constants are given elsewhere [18]. The PLP content of isolated H334G mutant was measured with a quantitative spectrophotometric test [46]. Role of His334 in a-glucan phosphorylase A. Schwarz et al. 5112 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS NMR spectroscopy All NMR measurements were recorded in 2 H 2 O (99.9% 2 H) at 30 °C on a Bruker (Rheinstetten, Germany) DRX 600 AVANCE spectrometer using topspin 1.3 soft- ware (Bruker). Proton, carbon and phosphorus spectra were measured at 600.13 MHz, 150.90 MHz, and 242.94 MHz, respectively. The one-dimensional spectra were recorded with 32 768 data points. Zero filling to 65 536 data points, appropriate exponential multiplication and Fourier transformation led to spectra with ranges of  5400 Hz ( 1 H),  33 000 Hz ( 13 C), and  24 000 Hz ( 31 P). pH-dependent 31 P chemical shifts were determined employ- ing a slight modification of a reported procedure [37]. Samples were prepared by adding 150 lL of 400–450 lm enzyme solution in 50 mm triethanolamine buffer, contain- ing 20 mm potassium sulfate, to 450 l L of a solution con- taining 50 mm triethanolamine buffer, 50 mm acetate and 20 mm potassium sulfate in 2 H 2 O. At the concentration used, sulfate does not inhibit the enzyme activity through competition with phosphate, suggesting that occupancy of the active site by sulfate is negligible. 31 P spectra were measured with proton decoupling and 2048 scans and a preacquisition delay of 1.0 s, resulting in spectra with a sig- nal-to-noise ratio of about 10 : 1 to 20 : 1 after about 1 h. Two-dimensional spectra were obtained from 256 experi- ments, each with 2048 data points and an appropriate num- ber of scans. Zero filling and Fourier transformation led to spectra with ranges of  5400 Hz and  30 000 Hz for proton and carbon, respectively. Chemical shifts have been referenced to external acetone (d H 2.225 p.p.m.; d C 31.45 p.p.m.) and 85% aqueous phosphoric acid (d P 0.00 p.p.m.). STD NMR spectra were measured employing a reported procedure [27]. Samples were prepared in 5 mm potassium phosphate buffer (pH 7.0), containing  10 lm enzyme as well as 5 mm ligand. Before addition to the NMR tube, the enzyme storage solution was gel-filtered using NAP 5 col- umns (GE Healthcare, Vienna, Austria) equilibrated with 5mm potassium phosphate buffer in 2 H 2 O (pH 6.65). Five hundred and twelve scans were collected, each with 50 Gaussian-shaped pulses (50 ms and 1 ms delay) and a 30 ms spin lock pulse, resulting in spectra of  4200 Hz spectral width. On and off resonance irradiations were made at d H ) 2.00 p.p.m. and d H 41.66 p.p.m., respectively, subtraction was performed via phase cycling, and no water suppression was applied. Reference proton spectra were recorded with 256 scans directly before and after the STD measurements. Acknowledgements Financial support from the Austrian Science Fund (FWF P15208-B09, P18038-B09 and P15118) is grate- fully acknowledged. References 1 Graves DJ & Wang JH (1972) a-Glucan phosphorylases ) chemical and physical basis of catalysis and regula- tion. In The Enzymes (Boyer PD, ed.), pp. 435–483. Academic Press, New York, NY. 2 Palm D, Klein HW, Schinzel R, Buehner M & Helmr- eich EJ (1990) The role of pyridoxal 5¢-phosphate in glycogen phosphorylase catalysis. Biochemistry 29, 1099–1107. 3 Madsen NB & Withers SG (1986) Pyridoxal phosphate and derivatives. In Coenzymes and Cofactors (Dolphin D, Paulson R & Avramovic O, eds), pp. 355–389. 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Eur J Biochem 185, 525–531. 37 Schinzel R, Palm D & Schnackerz KD (1992) Pyridoxal 5¢-phosphate as a 31 P reporter observing functional changes in the active site of Escherichia coli maltodex- trin phosphorylase after site-directed mutagenesis. Biochemistry 31, 4128–4133. 38 Becker S, Schnackerz KD & Schinzel R (1995) A study of binary complexes of Escherichia coli maltodextrin phosphorylase: a-d-glucose 1-methylenephosphonate as a probe of pyridoxal 5¢-phosphate–substrate interac- tions. Biochim Biophys Acta 1243, 381–385. 39 Klein HW & Helmreich EJ (1979) A proton donor– acceptor function of the 5¢-phosphate group of pyri- doxal-P in potato phosphorylase inferred from 31 P NMR spectra. FEBS Lett 108, 209–214. 40 Feldmann K & Hull WE (1977) 31 P nuclear magnetic resonance studies of glycogen phosphorylase from Role of His334 in a-glucan phosphorylase A. Schwarz et al. 5114 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... correlation between the reciprocal dissociation constants of GL binding to enzyme–phosphate complexes of wild-type CcStP and His334 mutants and kcat ⁄ Km values for phosphorolysis of maltopentaose (A) and starch (B) This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary... phosphorylase of bacterial a-glucan phosphorylases Biocatal Biotransform 19, 379–398 46 Griessler R, D’Auria S, Tanfani F & Nidetzky B (2000) Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein Protein Sci 9, 1149–1161 Supplementary material The following supplementary material... states of pyridoxal 5¢-phosphate Proc Natl Acad Sci USA 74, 856–860 Withers SG, Madsen NB & Sykes BD (1981) Active form of pyridoxal phosphate in glycogen phosphorylase Phosphorus-31 nuclear magentic resonance investigation Biochemistry 20, 1748–1756 Griessler R, Schwarz A, Mucha J & Nidetzky B (2003) Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. .. a-1,4-d-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering Biochem J 326, 773–783 Griessler R, Pickl M, D’Auria S, Tanfani F & Nidetzky B (2001) Oxyanion-mediated protein stabilization: differential roles of phosphate for preventing inactivation Role of His334 in a-glucan phosphorylase of bacterial... dimer structure of starch phosphorylase from Corynebacterium callunae Roles of Arg234 and Arg242 revealed by sequence analysis and site- directed mutagenesis Eur J Biochem 270, 2126–2136 Higuchi R, Krummel B & Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions Nucleic Acids Res 16, 7351–7367 Weinhausel A, Griessler... note: Blackwell Publishing is 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 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5115 . Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334 fi Gly mutant enzymes Alexandra. 40% that observed in the wild-type. Characterization of the H334G mutant Enzyme activity The H334G mutant exhibited 0.003% of the wild-type specific activity

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