Báo cáo khoa học: Catalytic residues Lys197 and Arg199 ofBacillus subtilis phosphoribosyl diphosphate synthase Alanine-scanning mutagenesis of the flexible catalytic loop

Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase

Tên đề tài: Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase

Tác giả: Bjarne Hove-Jensen, Ann-Kristin K. Bentsen and Kenneth W. Harlow

Lĩnh vực: Biological Chemistry, Institute of Molecular Biology and Physiology, University of Copenhagen, Denmark

Nội dung tài liệu: Nghiên cứu này tập trung vào việc phân tích vai trò của các gốc Lys197 và Arg199 trong enzyme phosphoribosyl diphosphate synthase (PRibPP synthase) của vi khuẩn Bacillus subtilis thông qua phương pháp đột biến alanine scanning. Các nhà nghiên cứu đã thay thế các codon mã hóa cho các axit amin trong vùng xúc tác linh hoạt của enzyme bằng codon cho alanine. Kết quả cho thấy sự đột biến ở Lys197 và Arg199 làm giảm đáng kể hoạt tính xúc tác của enzyme, với mức giảm Vmax lên đến hơn 30.000 lần đối với K197A và hơn 24.000 lần đối với R199A, trong khi ái lực với cơ chất (Km) ít bị ảnh hưởng. Điều này chỉ ra rằng Lys197 và Arg199 đóng vai trò quan trọng trong việc ổn định trạng thái chuyển tiếp của phản ứng xúc tác bởi PRibPP synthase. Nghiên cứu cũng xem xét các đột biến khác trong vùng xúc tác linh hoạt và cơ chế phản ứng của enzyme.

Mục lục chi tiết:

• Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase
• Alanine-scanning mutagenesis of the flexible catalytic loop
• Keywords
• Correspondence
• Present address
• Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197–208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine.
• The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity.
• The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail.
• The physical properties of the two enzymes were similar to those of the wildtype enzyme.
• Kinetic characterization showed that the Vmax values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced, respectively, compared to the wildtype enzyme.
• The Km values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged.
• Vapp values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the Vmax value of the wildtype enzyme.
• The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.
• The compound 5-phospho-D-ribosyl a-1-diphosphate (PRibPP) is an important component of the metabolism of most organisms.
• PRibPP is a precursor for the biosynthesis of purine, pyrimidine and pyridine nucleotides, as well as of the amino acids tryptophan and histidine [1,2].
• Microorganisms like Bacillus subtilis and Escherichia coli typically contain 10 enzymes that use PRibPP as a substrate [3].
• In addition, methanogenic archaea utilize PRibPP for the biosynthesis of methanopterin, a folate analogue involved in C1 metabolism [4], and Methanocaldococcus jannaschii utilizes PRibPP as a precursor of ribose 1,5-bisphosphate and subsequently, ribulose 1,5-bisphosphate [5].
• Finally, mycobacteria utilize PRibPP for the biosynthesis of polyprenylphosphate-pentoses [6].
• PRibPP is synthesized by transfer of the ẞ,y-diphosphoryl group of ATP to the C-1 hydroxyl of ribose 5-phosphate (Rib5P), in a reaction which is catalysed by PRibPP synthase (ATP:D-ribose 5-phosphate pyrophosphotransferase, EC 2.7.6.1) [7,8]: Rib5P + ATP → PRibPP + AMP.
• PRibPP synthase is encoded by the prs gene [9,10].
• Several crystal forms of B. subtilis PRibPP synthase have been obtained and the structure was solved to high resolution.
• The analysis revealed a two-domain subunit structure, which assembles to form a hexamer.
• Each domain contains a central five-stranded parallel ẞ-sheet surrounded by a-helices, and thus, the overall folds of the domains resemble those of type I phosphoribosyltransferases.
• Initially the flexible catalytic loop, KRRPRPNVAEVM(197–208), which contains several charged residues, remained unresolved, except for Lys197, Arg198 and Met208 [11].
• Among the amino acids of the loop, Lys197 and Arg199 are highly conserved, whereas the remaining 10 amino acids are only moderately conserved.
• Crystallization of the enzyme in the presence of the transition state analogue AlF3, the substrate Rib5P and the product AMP resulted in a structure with a bend arrangement of AMP-A1F3-AMP and with Rib5P attached in a manner that is believed to resemble the transition state with the exception of the addition of an adenosyl group [12].
• In this crystal form the flexible catalytic loop is fixed in a closed conformation that appears to shield the transition state analogue AlF3 from the solvent.
• Closure of the loop is stabilized by interaction of Lys197 through the ɛ-amino group and Arg199 through the guanidino group with two of the fluoride atoms, which are analogous to oxygen atoms of the ẞ-phosphorus of the substrate ATP.
• A transient negative charge that may develop on the ẞ-phosphoryl oxygen atoms could be stabilized by Lys197 and Arg199 [12].
• Furthermore, analysis of a crystal form with the inhibitor analogue a,ẞ-methylene GDP bound at the allosteric site as well as the substrate Rib5P and the reaction-inert substrate analogue α,β-methylene ATP present revealed a tight interface between two subunits.
• This interface is primarily formed by reciprocal interaction of hydrophobic amino acids of ẞ-strands located on either side of the flexible catalytic loop.
• The tightly packed interface prevents the closure of the loop, and thus, prevents the interaction of Lys197 and Arg199 with the phosphate chain of ATP.
• Release of this tight interaction following release of allosteric inhibitor binding causes a 7 Å displacement of the ẞ-strands, which is expected to allow the closure of the loop followed by catalysis.
• Closure of the loop is furthermore stabilized by hydrogen bonds formed between Asn203 and substrate-bound water molecules [12].
• Finally, a role of the flexible catalytic loop residue Arg198 in allosteric regulation was suggested.
• This arginine residue interacts primarily with Asp196 of the same subunit, but also with Asp186 of a neighbouring subunit, and in doing so assists in maintaining the tightly packed interface that prevents the closure of the flexible catalytic loop [12].
• The flexible catalytic loop of PRibPP synthase is topologically and functionally equivalent to a loop, variously designated the flexible loop, the catalytic loop, or loop II of the class I phosphoribosyltransferases.
• This loop is involved in the catalytic function of these enzymes by closing down on the bound substrates and thereby forming the active site (reviewed in [13,14]).
• Evidence for the importance of Lys197 in catalysis also comes from results of chemical modification of PRibPP synthase from E. coli with the substrate analogue 2′,3′-dialdehyde-ATP.
• Lysine residues 181, 193 and 230 were identified as possible reactive site residues.
• Of these, Lys193 is homologous to B. subtilis PRibPP synthase Lys197.
• It was suggested that this lysine residue might interact with the triphosphate chain of ATP either directly or indirectly through the Mg2+ ion chelated to the phosphate chain of the MgATP complex or it might form hydrogen bonds with the C-2′ or the C-3′ hydroxyl group of ATP [15].
• We report here the characterization of B. subtilis PRibPP synthase mutant forms with the individual amino acids of the flexible catalytic loop altered to alanine with emphasis on the two catalytic residues Lys197 and Arg199.
• Results
• Complementation of Aprs by mutant alleles specifying alanine substitutions of the flexible catalytic loop
• The codons of the flexible catalytic loop were altered individually to specify alanine as described in Experimental procedures.
• Each of the plasmids harbouring a prs-allele specifying an alanine variant was transformed to strain HO1088 (Aprs) and the growth of the transformants were analysed (Table 1).
• The results showed that the mutant alleles specifying K197A and R199A were unable to complement Aprs, as the transformants HO1088 (Aprs)/pAB700 (specifying K197A) and HO1088 (Aprs)/pHO377 (specifying R199A) grew only with all of the components of the PRibPP-consuming pathways present.
• This indicates the acquisition of PRibPP synthase with no or very low activity.
• In contrast, all of the remaining mutant alleles complemented the Aprs allele as the transformants grew without any of the compounds present.
• Kinetic analysis of K197A and R199A PRibPP synthases
• Each mutant variant enzyme was purified to near homogeneity, i.e., more than 98% purity as evaluated by SDS/PAGE.
• Their kinetic constants were determined together with those of wildtype PRibPP.
• Initial velocity vs the concentration of either ATP or Rib5P followed typical Michaelis-Menten kinetics.
• Kinetic parameters of the forward reaction of wildtype, K197A and R199A PRibPP synthases were obtained by measuring initial reaction rates under conditions where ATP and Rib5P were varied against each other.
• Double reciprocal plots of initial velocity vs ATP and fixed concentrations of Rib5P and vice versa showed a series of intersecting lines for both the wildtype enzyme, the K197A enzyme and the R199A enzyme.
• The K-values, obtained by fitting the data to Eqn (1), and assuming the binding of ATP before Rib5P (see below), are presented in Table 2.
• The two mutant enzymes displayed a large decrease in the maximal velocity.
• The Vmax value of the K197A enzyme was more than 30 000-fold reduced compared to that of the wildtype enzyme, whereas that of the R199A enzyme was more than 24 000-fold reduced.
• In contrast, KATP and Krib5p values of the mutant enzymes were much less altered, if at all, compared to those of the wildtype enzyme.
• The Ki(ATP) value of the K197A mutant was reduced six-fold compared to that of the wildtype enzyme, whereas that of the R199A enzyme was three-fold reduced.
• Inhibition of K197A, R198A and R199A PRibPP synthases
• The mode of inhibition by ADP of wildtype PRibPP synthase was analysed.
• Double reciprocal plots of activity vs ATP concentration at fixed concentration of ADP were used to calculate values of slopes and intercepts.
• Fitting of the slope and intercept values as parabolic inhibition [16] failed to give a reasonable description of the data.
• Instead the data were fitted to Eqn (5).
• Replots of intercepts and slopes revealed parabolic curves which are characteristic for binding of ADP to both an active site and to an independent, allosteric site of PRibPP synthase [17,18].
• A similar analysis was performed with the K197A, R199A, and R198A mutant enzymes.
• The Arg198 residue has been proposed to be involved in allosteric regulation due to its involvement in subunit-subunit interactions [12].
• The inhibition constants obtained from this analysis are given in Table 3.
• Although the inhibition pattern appeared similar for the wildtype, the R198A and the R199A enzyme species, i.e., the inhibition by ADP was nonlinear noncompetitive with respect to ATP, the inhibition of the K197A mutant PRibPP synthase was altered.
• The K’is value of the K197A mutant was only three-fold higher than that of the wildtype enzyme, but the K’ii value of the mutant enzyme was 12-fold higher than that of the wildtype enzyme.
• The apparent Hill coefficient n indicates that ADP binds to nonidentical sites on the enzyme, consistent with ADP binding both to the active and the allosteric site.
• From the data it appears that the cooperativity of ADP binding is reduced for the K197A mutant enzyme.
• Stability of K197A and R199A PRibPP synthases
• To see if the mutations had an influence on the quaternary structure, the K197A and R199A mutant as well as wildtype PRibPP synthases were subjected to electrophoresis in nondenaturing gels.
• All three PRibPP synthases migrated as single species, with a slight increase in mobility of the K197A enzyme relative to that of the wildtype enzyme, and with a slight increase in mobility of the R199A enzyme relative to that of the K197A enzyme.
• This is the result expected from the loss of six positively charged lysine or arginine residues per hexamer (data not shown).
• In addition, the temperature of irreversible denaturation of the three enzymes was analysed by differential scanning calorimetry.
• This analysis revealed that the major transition temperature of the wildtype and the two mutant enzymes was quite similar, as the apparent transition temperature was determined as 62.8 °C for the wildtype enzyme, 61.2 °C for the K197A enzyme, and 62.6 °C for the R199A enzyme (data not shown).
• Altogether the results of native gel electrophoresis and differential scanning calorimetry show that the presence of alanine rather than lysine at position 197, or alanine rather than arginine at position 199 appeared to have no effect on either structure or stability of the enzyme.
• Kinetic analysis of nine other mutants of the flexible catalytic loop
• The remaining mutants were less thoroughly analysed.
• Values of Vapp as well as Km for ATP were determined by varying the ATP concentration at a fixed Rib5P concentration (Table 4).
• In general the Vapp values were reduced compared to the Vmax value of the wildtype enzyme.
• However, the reduction was much less severe than that determined for the K197A or R199A mutant enzymes.
• The Vapp values ranged between 20 and 100% of the Vmax value of the wildtype enzyme.
• The R201A, N203A, E206A and M208A enzymes had Vapp values similar to the Vmax value of the wildtype.
• The Km values for ATP varied somewhat, but the values were not significantly different from the KATP value of the wildtype enzyme reported above, with the exception of the R201A, N203A and E206A enzymes, which revealed an approximate 3.0-, 4.5- and 2.5-fold increase, respectively, in apparent Km.
• Reaction mechanism of B. subtilis PRibPP synthase
• As mentioned above, the double reciprocal plots of initial velocity vs ATP and fixed concentrations of Rib5P or vice versa showed intersecting lines, which indicated a sequential mechanism.
• To determine if the binding of the substrates was ordered or random, product inhibition was analysed.
• Measurements were made with both of the products, PRibPP and AMP, varied against different concentrations of ATP at fixed Rib5P concentrations and vice versa.
• Inhibition by AMP was noncompetitive with respect to Rib5P and competitive with respect to ATP.
• Inhibition by PRibPP was competitive with respect to ATP and noncompetitive with respect to Rib5P.
• The calculated inhibition constants are given in Table 5.
• These are the results predicted for an ordered binding of the substrates with ATP binding first and a random release of the products [16].
• Discussion
• The kinetic analysis of the K197A and R199A mutant PRibPP synthases indicates that these amino acids are vitally important for catalysis because more than 30 000- and 24 000-fold reductions in Vmax values were determined in the absence of significant effects on substrate binding, respectively.
• The KATP and Krib5P values of both mutant enzymes were similar to those of the wildtype PRibPP synthase.
• These results strongly support the hypothesis that Lys197 and Arg199 are involved in catalysis by interacting with the ẞ-phosphorus oxygen atoms of the ATP triphosphate chain after formation of the enzyme-substrate complex.
• According to this hypothesis, this interaction results in