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• W2056810020 abstract "Kinetic as well as chemical modification studies have implicated the presence of an active site arginine in choline acetyltransferase, whose function is to stabilize coenzyme binding by interacting with the 3′-phosphate of the coenzyme A substrate. In order to identify this residue seven conserved arginines in rat choline acetyltransferase were converted to alanine by site-directed mutagenesis, and the properties of these mutants were compared with the wild type enzyme. Substitution of arginine 452 with alanine resulted in a 7-12-fold increase in the Km for both CoA and acetylcholine as well as kcat, with little change in the Km for dephospho-CoA. Product inhibition studies showed choline to be a competitive inhibitor with respect to acetylcholine, indicating R452A follows the same Theorell-Chance kinetic mechanism as the wild type enzyme. Similar results were obtained with R452Q and R452E, with the latter showing the largest changes in kinetic parameters. These findings are consistent with Arg-452 mutations increasing the rate constant, k5, for dissociation of the coenzyme from the enzyme. Direct evidence that arginine 452 is involved in coenzyme A binding was obtained by showing a 5-10-fold decrease in affinity of the R452A mutant for coenzyme A as determined by the ability to protect against phenylglyoxal inactivation as well as thermal inactivation. Kinetic as well as chemical modification studies have implicated the presence of an active site arginine in choline acetyltransferase, whose function is to stabilize coenzyme binding by interacting with the 3′-phosphate of the coenzyme A substrate. In order to identify this residue seven conserved arginines in rat choline acetyltransferase were converted to alanine by site-directed mutagenesis, and the properties of these mutants were compared with the wild type enzyme. Substitution of arginine 452 with alanine resulted in a 7-12-fold increase in the Km for both CoA and acetylcholine as well as kcat, with little change in the Km for dephospho-CoA. Product inhibition studies showed choline to be a competitive inhibitor with respect to acetylcholine, indicating R452A follows the same Theorell-Chance kinetic mechanism as the wild type enzyme. Similar results were obtained with R452Q and R452E, with the latter showing the largest changes in kinetic parameters. These findings are consistent with Arg-452 mutations increasing the rate constant, k5, for dissociation of the coenzyme from the enzyme. Direct evidence that arginine 452 is involved in coenzyme A binding was obtained by showing a 5-10-fold decrease in affinity of the R452A mutant for coenzyme A as determined by the ability to protect against phenylglyoxal inactivation as well as thermal inactivation. INTRODUCTIONThe enzyme choline acetyltransferase (ChAT, 1The abbreviations used are: ChATcholine acetyltransferaseChcholinePAGEpolyacrylamide gel electrophoresis. EC 2.3.1.6) catalyzes the transfer of the acyl group from acetyl-CoA to choline resulting in the formation of the neurotransmitter acetylcholine. Mechanistic studies on the enzyme suggest a concerted reaction in contrast to similar enzymes that transfer acyl groups via an acyl enzyme intermediate. The kinetic mechanism for the enzyme approximates a Theorell-Chance mechanism(1Tucek S. Whittaker V.P. Handbook of Experimental Pharmacology. 86. Springer-Verlag, Berlin1988: 129-131Google Scholar), although by using isotope exchange at equilibrium a random component in the reaction has been detected(2Hersh L.B. J. Biol. Chem. 1982; 257: 12820-12825Google Scholar). Chemical modification studies have implicated histidine, cysteine, and arginine as active site residues. Thus inactivation studies with dithiobis-4-nitro-2-carboxylate led to the proposal that the enzyme contains an active site cysteine(3Roskoski Jr., R. J. Biol. Chem. 1974; 249: 2156-2159Google Scholar, 4Roskoski Jr., R. Lim C.T. Roskoski L.M. Biochemistry. 1975; 14: 5105-5110Google Scholar, 5Driskell W.J. Weber B.H. Roberts E. J. Neurochem. 1978; 30: 1135-1141Google Scholar, 6Hersh L.B. J. Neurochem. 1979; 32: 991-996Google Scholar, 7Carbini L. Rodriguez G. Hersh L.B. Brain Res. Bull. 1990; 24: 119-124Google Scholar); however, modification of this residue by methylation showed it is not essential for catalysis(8Hersh L.B. Nair R.V. Smith D.J. J. Biol. Chem. 1979; 254: 11988-11992Google Scholar). Similarly, an active site histidine was implicated by inactivation studies with diethylpyrocarbonate while an active site arginine was implicated by inactivation studies with phenylglyoxal(7Carbini L. Rodriguez G. Hersh L.B. Brain Res. Bull. 1990; 24: 119-124Google Scholar, 9Mautner H.G. Pakula A.A. Merrill R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7449-7452Google Scholar). It has been suggested that the active site histidine serves as a general acid/base catalyst(10Currier S.F. Mautner H.G. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3355-3358Google Scholar), while the active site arginine is postulated to be involved in binding interactions with the 3′-phosphate of the substrate CoA(9Mautner H.G. Pakula A.A. Merrill R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7449-7452Google Scholar).With the availability of cDNA clones for ChAT from Drosophila melanogaster(11Itoh N. Slemmon J.R. Hawke D.H. Williamson R. Morita E. Itakura K. Roberts E. Shively J.E. Crawford G.D. Salvaterra P.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4081-4085Google Scholar), porcine spinal cord(12Berrard S. Brice A. Lottspeich F. Braun A. Barde Y.A. Mallet J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9280-9284Google Scholar), rat brain(13Brice A. Berrard S. Raynaud B. Ansieau S. Coppola T. Weber M.J. Mallet J. J. Neurosci. Res. 1989; 23: 266-273Google Scholar, 14Ishii K. Oda Y. Ichikawa T. Deguchi T. Mol. Brain Res. 1990; 7: 151-159Google Scholar), mouse brain(14Ishii K. Oda Y. Ichikawa T. Deguchi T. Mol. Brain Res. 1990; 7: 151-159Google Scholar), and Caenorhabditis elegans(15Alfonso A. Grundahl K. McManus J.R. Rand J.B. J. Neurosci. 1994; 14: 2290-2300Google Scholar) it has now become possible to use site-directed mutagenesis to identify these active site residues and to study their function. We have previously used site-directed mutagenesis to analyze the functionality of three conserved histidines in Drosophila ChAT and have shown that one of these residues, His-426, is essential for catalysis(16Carbini L.A. Hersh L.B. J. Neurochem. 1993; 61: 247-253Google Scholar). We have now used a similar approach to search for the active site arginine thought to be involved in coenzyme A binding. Seven conserved arginines were individually changed to alanine and the resultant mutants characterized. The properties of mutant enzymes containing substitutions at arginine 452 are consistent with this arginine serving as an active site residue.EXPERIMENTAL PROCEDURESMaterialsCitrate synthase and malate dehydrogenase, both from pig heart, were obtained from Sigma. Ni-nitriloacetate-agarose was obtained from Qiagen, while Immobilon-P nylon membranes were obtained from Millipore Corp. A rabbit anti-human ChAT antiserum prepared as described previously was employed for Western blot analyses. Alkaline phosphatase conjugated to goat anti-rabbit IgG was obtained from Bio-Rad.Site-directed MutagenesisOligonucleotide-directed mismatch mutagenesis was performed by the “dual primer” method of Zoller and Smith(17Zoller M.J. Smith M. DNA. 1984; 3: 479-484Google Scholar). The conserved arginine residues and the changes that were made in them are listed in Table 1. The mutated cDNA fragments were inserted into the expression vector pQE9 as described(18Wu D. Ahmed S.N. Lian W. Hersh L.B. J. Biol. Chem. 1995; 270: 19395-19401Google Scholar), and final confirmation of the nucleotide changes in the clones was achieved by dideoxy chain-terminating DNA sequencing using Sequenase version 2.0 from U. S. Biochemical Corp.TABLE I Open table in a new tab Expression and Purification of Recombinant ChATEscherichia coli SG12036 expressing recombinant rat ChAT was grown at 22°C for 4 days in LB media. Cells were harvested by centrifugation and frozen at -80°C until further use. Enzyme purification was achieved as described previously(18Wu D. Ahmed S.N. Lian W. Hersh L.B. J. Biol. Chem. 1995; 270: 19395-19401Google Scholar). This procedure involves application of an E. coli extract, prepared in 20 mM sodium phosphate buffer, pH 7.6, containing the recombinant enzyme on a column of Ni-nitriloacetate-agarose equilibrated with the extraction buffer. The column is washed batchwise with buffer containing increasing concentrations of NaCl from 0 to 2 M and lastly with buffer containing 5 mM imidazole, pH 7.4. The enzyme, eluted with an imidazole gradient from 5 to 150 mM, was applied to a column of blue-agarose (Amicon), previously equilibrated with 20 mM sodium phosphate buffer, pH 7.6. After washing the column with equilibration buffer, the enzyme was eluted with a linear salt gradient from 0 to 1 M and concentrated/dialyzed in a Centricon 30 concentrator (Amicon). The purified enzyme was either used immediately or stored at -80°C in 20 mM sodium phosphate buffer, pH 7.6, containing 40% of glycerol.Assay of ChAT ActivityEnzymatic activity was determined by the fluorometric assay of Hersh et al.(19Hersh L.B. Coe B. Casey L. J. Neurochem. 1978; 30: 1077-1085Google Scholar). This assay involves coupling the reverse ChAT reaction (CoA + AcCh → AcCoA + Ch) to citrate synthase and malate dehydrogenase. Assays conducted under high salt conditions contained 10 mM potassium phosphate buffer (pH 7.4), 250 mM sodium chloride, 0.125 mM NAD, 0.5 mML-malate, 0.1 mMDL-dithiothreitol, 1.5 units of pig heart citrate synthase, 4 units of pig heart malate dehydrogenase, and variable levels of acetylcholine chloride and CoA. For routine assays 25 mM acetylcholine chloride and 0.1 mM CoA were used. Reactions were initiated by the addition of acetylcholine chloride, and NADH formation was monitored continuously using an Optical Technologies fluorometer. Assays conducted under low salt conditions were identical except that NaCl was omitted from the reaction.Characterization of Purified ChATEnzyme purity was monitored by SDS-PAGE(20Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar), with the protein stained by the alkaline silver staining procedure(21Switzer R.C. Merril C.R. Shifrin S. Anal. Biochem. 1979; 98: 231-237Google Scholar). The concentration of purified ChAT was determined by the Bradford method (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as standard. For R250A, Western blot analysis was used to quantitate the amount of enzyme using known amounts of purified wild type enzyme as a standard. Briefly, proteins were electrophoretically transferred to an Immobilon-P nylon membrane as described by Towbin et al.(23Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar). After transfer, the membranes were incubated for 1 h in blocking buffer (10 mM Tris-HCl buffer, pH 7.4, 150 mM sodium chloride, 5% nonfat dry milk, and 0.2% Nonidet P-40) and then overnight with affinity-purified anti-ChAT antibodies, diluted into fresh blocking buffer. The following morning the membrane was washed three times with 10 mM Tris-HCl buffer (pH 7.4), 150 mM sodium chloride, 0.25% sodium 7-deoxycholate, 0.1% SDS, followed by another three washes with 10 mM Tris-HCl (pH 7.4) buffer containing 150 mM NaCl. To visualize the immunoreactive protein a goat anti-rabbit antiserum coupled with alkaline phosphatase was employed. 5-Bromo-4-chloro-3-indolyl phosphate coupled with p-nitroblue tetrazolium chloride were used as the color reagents.Determination of Kinetic ConstantsPreliminary estimates of the Km values for CoA, dephospho-CoA, and acetylcholine were made with each mutant at substrate concentrations saturating (10 × Km) for the wild type enzyme. Where the Km appeared higher than the wild type enzyme, the kinetic measurements were redetermined at a higher fixed substrate concentration to ensure saturation. This protocol could not be used for determining the Km of CoA and dephospho-CoA under low salt conditions with the R452A mutant since the concentration of acetylcholine, used as its chloride salt, needed to achieve a saturating level would have raised the anion concentration into the range used at high salt. Thus to determine the Km for CoA and dephospho-CoA with this mutant under low salt conditions, the concentration of acetylcholine was varied at fixed variable levels of CoA or dephospho-CoA, maintaining the concentration of acetylcholine below 10 mM. The data were then replotted as 1/Vmax (equivalent to an infinite acetylcholine concentration) versus 1/CoA (or dephospho-CoA). Kinetic constants were determined by fitting the data to the weighted least squares kinetic programs of Cleland. The same Vmax was obtained with either substrate indicating saturation was indeed achieved.Chemical Modification of ChAT by PhenylglyoxalEnzyme was incubated at 37°C with 5 mM phenylglyoxal in 20 mM sodium phosphate buffer, pH 7.6, containing 0.25 M NaCl and 1 mg/ml bovine serum albumin. The reaction was initiated by the addition of phenylglyoxal, and aliquots of 6 μl were withdrawn at various times and added to an assay mixture containing 10 mM sodium phosphate buffer, pH 7.6, 10 mM choline iodide, and 10 mM radiolabeled acetyl-CoA in a total volume of 50 μl. Acetylcholine formation was measured by the method of Fonnum (24Fonnum F. J. Neurochem. 1975; 24: 407-409Google Scholar).Heat Inactivation of ChAT and Substrate ProtectionEnzyme was incubated at the desired temperature in 20 mM sodium phosphate buffer, pH 7.6, in the presence or absence of CoA at the indicated concentration. Aliquots were withdrawn at various times, diluted with an equal volume of ice-cold 20 mM sodium phosphate buffer, pH 7.6, containing 2 mg/ml bovine serum albumin. The samples were maintained on ice until assayed by the standard fluorometric assay.RESULTSPrevious studies have implicated an active site arginine residue to be involved in the binding of coenzyme A to the enzyme ChAT. Comparison of the amino acid sequences of ChAT from rat, Drosophila, and C. elegans, deduced from their respective cDNAs, revealed seven potential conserved arginines (Table 1). In order to determine which, if any, are involved in coenzyme binding we utilized a rat ChAT cDNA and alanine scanning in which each of these putative active site arginines was separately changed to an alanine by site-directed mutagenesis. Each cDNA was constructed in an expression vector containing an N-terminal hexahistidine fused to the ChAT protein. Previous studies (18Wu D. Ahmed S.N. Lian W. Hersh L.B. J. Biol. Chem. 1995; 270: 19395-19401Google Scholar) have established that this modification has no effect on the kinetic properties of the enzyme. Expression of the recombinant enzymes in E. coli, in all but one case, led to their facile purification by a two-step procedure involving metal affinity chromatography followed by dye binding chromatography as described under “Experimental Procedures.”Fig. 1 shows representative preparations of the purified enzymes in which it can be seen that essentially homogeneous enzyme is obtained. In one case, the conversion of arginine 99 to alanine resulted in low levels of expression of this mutant, and difficulties were subsequently encountered in purifying this recombinant protein. Therefore, the properties of this mutant were analyzed in E. coli extracts rather than with purified enzyme.The kinetic properties of the mutant enzymes were initially compared with the wild type enzyme in the presence of 0.25 M NaCl, conditions previously shown to give maximal activity(25Hersh L.B. Peet M. J. Neurochem. 1978; 30: 1087-1093Google Scholar). The results of this kinetic analysis are summarized in Table 2. Comparing the various alanine substitutions, the most significant effect observed is with the mutant R452A in which the Km for CoA increases more than 12-fold, the Km for acetylcholine increases ~8-fold, and kcat increases ~7-fold. Smaller increases in the Km for CoA and acetylcholine were observed with the mutants R453A, R458A, and R463A. These studies were further extended by replacing arginine 452 with glutamine, which is a near isosteric replacement, and also with glutamate, which produces a reversal in charge at this position. The kinetic properties of the R452Q mutant were essentially the same as the R452A mutant, while the R452E mutant showed greater increases in the Km for CoA (54-fold), the Km for acetylcholine (60-fold), and kcat (~15-fold) (Table 2). We also tested the effect of charge reversal on the adjacent arginine, arginine 453, and the effect of replacing both arginines 452 and 453 with glutamine. Charge reversal at position 453 produced an enzyme form similar to R452A or R452Q, while replacing both arginines produced an enzyme form in which the Km for CoA increased more than 170-fold, the Km for acetylcholine increased ~70-fold, and kcat increased ~17-fold.TABLE II Open table in a new tab Since it has been proposed that an active site arginine functions to interact with the 3′-phosphate of coenzyme A(9Mautner H.G. Pakula A.A. Merrill R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7449-7452Google Scholar), the effect of each mutation with dephospho-CoA as substrate was examined, since no such interaction should occur with this substrate. With the wild type enzyme dephospho-CoA exhibits a Km that is ~10-fold higher than coenzyme A (Table 2). In each of the mutants the Km for dephospho-CoA was similar to that obtained with the wild type enzyme, this being particularly notable with R452E and R452Q/R453Q in which the Km for dephospho-CoA increased less than 4-fold as compared with changes in the Km for CoA ~of more than 50-fold for these mutants. Thus at this initial level of analysis Arg-452 appears to be a likely candidate as an active site residue, with arginines 453, 458, and 463 also being possible candidates.It has been previously observed that anions affect the kinetic parameters of the ChAT reaction(26Rossier J. Int. Rev. Neurobiol. 1977; 20: 284-337Google Scholar). That is, anions act to increase Vmax but at the same time increase the Km for both substrates(25Hersh L.B. Peet M. J. Neurochem. 1978; 30: 1087-1093Google Scholar). It has been suggested that this effect can be attributed, at least in part, to anions interfering with the binding of the 3′-phosphate of coenzyme A to an active site arginine(9Mautner H.G. Pakula A.A. Merrill R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7449-7452Google Scholar). Thus a similar kinetic analysis was conducted with the wild type enzyme and the four candidate active site arginine mutants under conditions of low ionic strength. The results of this analysis are shown in Table 3. In agreement with previous studies(25Hersh L.B. Peet M. J. Neurochem. 1978; 30: 1087-1093Google Scholar, 26Rossier J. Int. Rev. Neurobiol. 1977; 20: 284-337Google Scholar), it can be seen that with the wild type enzyme decreasing the anion concentration decreases the Km for CoA 12-fold but decreases the Km for choline less than 3-fold and lowers kcat by approximately 2-fold. In contrast decreasing salt has little effect on the Km for dephospho-CoA, a finding consistent with the proposal that anions compete for the interaction at the 3′-phosphate of coenzyme A with an arginine.TABLE III Open table in a new tab The effects of decreased ionic strength are different with the R452A mutant. The Km for CoA is reduced only ~3-fold by lowering the ionic strength of the assay, while there is little change in the Km for acetylcholine and kcat is essentially unchanged. As with the wild type enzyme the Km for dephospho-CoA is barely affected by changes in anion concentration. The other putative active site arginine mutants all exhibit changes in their kinetic properties that are similar to those observed with the wild type enzyme except that kcat remained unchanged. Again these results are consistent with arginine 452 as the active site arginine interacting with CoA.In order to test for a change in kinetic mechanism between the wild type enzyme and the R452A mutant we determined the inhibition pattern with choline as a product inhibitor. The ChAT reaction follows primarily a Theorell-Chance kinetic mechanism, characterized by competitive inhibition between the inner substrate pair choline and acetylcholine(25Hersh L.B. Peet M. J. Neurochem. 1978; 30: 1087-1093Google Scholar). As shown in Fig. 2, choline acts as a competitive inhibitor with respect to acetylcholine for both the wild type enzyme and for the R452A mutant, a finding suggesting that both enzymes exhibit the same Theorell-Chance kinetic mechanism. However, the Ki for choline was greater for the mutant (1.1 mM) as compared with the wild type enzyme (0.1 mM). Although not shown, competitive inhibition between choline and acetylcholine was also observed with the most severely affected mutant R452Q/R453Q with the Ki for choline increased to 12 mM.Figure 2:Choline as a competitive inhibitor of wild type ChAT and R452A. Choline inhibition was determined by the fluorometric assay in the presence of 0.25 M NaCl, variable acetylcholine, and a fixed level of CoA (2 × Km(CoA)). Data were plotted as 1/v versus 1/[AcCh]. Ki was calculated from the equation for a competitive inhibitor, v = Vmax (Km/[AcCh]{1 + [I]/Ki} + 1). Choline inhibition of wild type enzyme: ■, no added choline; □, 0.1 mM choline; •, 0.3 mM choline. Choline inhibition of R452A mutant: ■, no added choline; □, 1.0 mM choline; •, 3.0 mM choline.View Large Image Figure ViewerDownload (PPT)We next analyzed the ability of each of the alanine mutants to react with the arginine-specific reagent phenylglyoxal. As shown in Fig. 3 treatment of the wild type enzyme with 5 mM phenylglyoxal results in biphasic inactivation. There is a rapid initial decline of activity, which is too fast to measure but which results in a loss of ~35% enzyme activity. This is followed by a slower loss in enzyme activity, which plateaus at ~90% inactivation. A replot of the data as log (% activity remaining) versus time from 0.1 to 45 min is linear assuming an end point of 90% inactivation. From this analysis a half-time of ~11 min was obtained. As shown in Fig. 3 the slow phase of inactivation was prevented by inclusion of acetyl-CoA in the inactivation reaction at a concentration in the range of the Km for CoA. Acetylcholine was without effect. Inclusion of acetyl-CoA at concentrations greater than 50 times the Km for CoA had no effect on the fast phase (data not shown). Conversion of arginines 99, 312, and 453 to alanine had no significant effect on phenylglyoxal inactivation. In each of these cases the same extent of enzyme inactivation was observed in the rapid phase, and the half-time for the slow phase varied only slightly from that observed with the wild type enzyme (10, 12, and 10 min for alanine substitutions at arginines 99, 312, and 453, respectively).Figure 3:Phenylglyoxal inactivation of wild type and Arg → Ala mutants of rat ChAT. Phenylglyoxal inactivation was conducted as described under “Experimental Procedures” in the presence of 5 mM phenylglyoxal and when added acetylcholine or acetyl-CoA. In each case inactivation in the presence of 5 mM phenylglyoxal is designated as the open square while the control incubation in the absence of phenylglyoxal is given by the filled square. Upper left panel, inactivation of wild type enzyme as noted and in the presence of 1.8 mM acetylcholine (•) or 0.5 μM acetyl-CoA (▵); upper right panel, inactivation of R250A; lower left panel, inactivation of R452A as noted and in the presence of 0.5 μM acetyl-CoA (▵) or 13.6 μM acetyl-CoA (▲); lower right panel, inactivation of R463A as noted and in the presence of 2.5 μM acetyl-CoA (▲).View Large Image Figure ViewerDownload (PPT)Changing arginine 452 to alanine resulted in a biphasic inactivation curve similar to the wild type enzyme except that the secondary phase was faster (t0.5~7 min) and inactivation went to completion Fig. 3. Acetyl-CoA at the same concentration used to protect the wild type enzyme from inactivation was ineffective with this mutant; however, increasing the acetyl-CoA concentration to 13.6 μM was able to afford protection (Fig. 3). Although not shown, acetylcholine at 15 mM had no effect on phenylglyoxal inactivation.Changing arginine 463 to Ala resulted in a considerably more rapid rate of inactivation by phenylglyoxal (Fig. 3). In this case the initial rapid and secondary phases of the reaction could not be distinguished. However, using 2.5 μM acetyl-CoA to protect against inactivation, the biphasic nature of the inactivation process became apparent (Fig. 3). Changing Arg-250 to Ala totally eliminated the secondary phase of inactivation by phenylglyoxal (Fig. 3).In order to provide additional evidence that Arg-452 is involved in CoA binding, we measured the ability of coenzyme A to protect the enzyme against thermal inactivation. The wild type enzyme is inactivated at 48°C under low ionic strength conditions with a t0.5 of 60 s. As shown in Fig. 4, CoA afforded partial protection against thermal inactivation exhibiting a Kd value of ~0.4 μM, a value similar to the kinetic Km of 0.25 μM listed in Table 2. The R452A mutant was more thermolabile being inactivated at 48°C with a t0.5 of ~20 s. At 44°C the t0.5 was 35 s, and CoA also provided partial protection against thermal inactivation; however, in this case the estimated binding constant was shifted to ~6 μM.Figure 4:CoA protection of thermal inactivation of wild type ChAT and R452A. Left, wild type enzyme inactivated at 48°C in the presence of the indicated concentration of CoA. Half-times were obtained from plots of log activity remaining versus time at each CoA concentration. Right, R452A inactivated at 44°C in the presence of the indicated concentration of CoA. Half-times were obtained from plots of log activity remaining versus time at each CoA concentration.View Large Image Figure ViewerDownload (PPT)DISCUSSIONPrevious studies have shown that the enzyme choline acetyltransferase is inactivated by arginine-specific reagents in a reaction protected by the substrate acetyl-CoA(9Mautner H.G. Pakula A.A. Merrill R.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7449-7452Google Scholar). This observation, in conjunction with the results of kinetic studies which showed that dephospho-coenzyme A was poorly bound by the enzyme(6Hersh L.B. J. Neurochem. 1979; 32: 991-996Google Scholar), led to the proposal of an active site arginine that interacts with the 3′-phosphate of coenzyme A. Since the results of chemical modification experiments are often equivocal or ambiguous, we utilized alanine scanning to systematically search for this active site arginine by replacing seven conserved arginines with alanine. The results of the kinetic analysis of these mutant enzymes are consistent with arginine 452 being the likely candidate. The predicted properties of such a mutant would include a decreased affinity for CoA with little change in the affinity of the mutant for dephospho-CoA. In accordance with this predicted behavior conversion of arginine 452 to alanine, glutamine, or even glutamate caused a 12-50-fold increase in the Km for CoA but less than a 3-fold change in the Km for dephospho-CoA. Thus in contrast to the wild type enzyme, where the Km for CoA is ~10-fold lower than the Km for dephospho-CoA, the Km values for CoA and dephospho-CoA are nearly the same in R452A and R452Q. With R452E dephospho-CoA becomes a better substrate than CoA. The observation that the properties of R452A and R452Q are quite similar rules out any complications that might have been introduced as a result of a change in the side chain volume when substituting the non-isosteric alanine for arginine. Thus this data is consistent with arginine 452 interacting with the 3′-phosphate of coenzyme A. The effects of changing arginine 452 by mutagenesis may be blunted by the presence of an adjacent arginine in position 453. Arginine 453 may also be directly involving in coenzyme binding or alternatively may realign in the Arg-452 mutants so that it can participate in coenzyme binding. This might explain why the most dramatic effects are seen with the R452Q/R453Q double mutant in which both arginines have been substituted.At first glance it might seem surprising that the Km for both CoA and choline as well as kcat would be increased by mutations at position 452. In the Theorell-Chance kinetic mechanism, which the Arg-452 mutant appears to obey, the rate-limiting step in the reaction is the release of product from the enzyme, k5 in Fig. S1. The Km values for coenzyme A and acetylcholine reflect their respective affinity in the steady state and are defined as the ratio of k5/k1 and k5/k3, respectively. The data are consistent with mutations at Arg-452 decreasing the affinity of the enzyme for CoA (or acetyl-CoA) by loss of the interaction with the 3′-phosphate. Kinetically this is manifested as an increase in the rate constant k5 (kcat), which represents dissociation of the coenzyme from the enzyme. An increase in k5 would also be expected to result in an increase in Km(acetylcholine) since this kinetic constant is directly proportional to k5. With R452A and R452Q there is a constant 7-12-fold increase in kcat, Km(acetylcholine), and Km(CoA), a f" @default.
• W2056810020 created "2016-06-24" @default.
• W2056810020 creator A5000098648 @default.
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• W2056810020 date "1995-12-01" @default.
• W2056810020 modified "2023-09-27" @default.
• W2056810020 title "Identification of an Active Site Arginine in Rat Choline Acetyltransferase by Alanine Scanning Mutagenesis" @default.
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• W2056810020 doi "https://doi.org/10.1074/jbc.270.49.29111" @default.
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