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• W2062876845 abstract "Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N -acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent p Ka of 7.3) contributes ∼6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes ∼150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) p Ka of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors. Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N -acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent p Ka of 7.3) contributes ∼6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes ∼150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) p Ka of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone produced in the brain by the pineal gland and controls behavioral and physiological circadian rhythms. The production of this hormone is dependent on the enzyme serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT) 1The abbreviations used are: AANATarylalkylamine N-acetyltransferase (serotonin N-acetyltransferase)MES2-(N-morpholino)ethanesulfonic acidMOPS3-(N-morpholino)propanesulfonic acidEPPSN-2-hydroxyethylpiperazine-N′-3-propanesulfonic acidCHES2-(N-cyclohexylamino)ethanesulfonic acidGSTglutathione S-transferaser.m.s.d.root mean square difference ), which fluctuates in response to light and dark signals and is the rate-limiting enzyme in the biosynthesis pathway (see Fig. 1 A) (1Klein D.C. Coon S.L. Roseboom P.H. Weller J.L. Bernard M. Gastel J.A. Zatz M. Iuvone P.M. Rodriquez I.R. Begay V. Falcon J. Cahill G.M. Cassone V.M. Baler R. Recent Prog. Horm. Res. 1997; 52: 307-357PubMed Google Scholar). Concurrent with the fluctuation in AANAT, the level of circulating melatonin is high at night and low during the day (1Klein D.C. Coon S.L. Roseboom P.H. Weller J.L. Bernard M. Gastel J.A. Zatz M. Iuvone P.M. Rodriquez I.R. Begay V. Falcon J. Cahill G.M. Cassone V.M. Baler R. Recent Prog. Horm. Res. 1997; 52: 307-357PubMed Google Scholar). Upon sudden exposure to light in the middle of the night, levels of melatonin and AANAT decrease rapidly with a half-life of ∼3.5 min (2Gastel J.A. Roseboom P.H. Rinaldi P.A. Weller J.L. Klein D.C. Science. 1998; 279: 1358-1360Crossref PubMed Scopus (255) Google Scholar). arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase) 2-(N-morpholino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid 2-(N-cyclohexylamino)ethanesulfonic acid glutathione S-transferase root mean square difference AANAT is a member of the GNAT (GCN-5-related N -acetyltransferase) superfamily of enzymes, the other members of which include GCN-5, PCAF (p300/CBP-associatedfactor), and the aminoglycoside N-acetyltransferases. This superfamily has members found in all kingdoms of life and is characterized by a common substrate, acetyl-CoA, and a structural fold where acetyl-CoA binds (3Neuwald A.F. Landsman D. Trends Biochem. Sci. 1997; 22: 154-155Abstract Full Text PDF PubMed Scopus (387) Google Scholar). The primary sequences of these enzymes are poorly conserved, reflecting their ability to acetylate a variety of different substrates from histones to aminoglycoside antibiotics (3Neuwald A.F. Landsman D. Trends Biochem. Sci. 1997; 22: 154-155Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 4Marmorstein R. J. Mol. Biol. 2001; 311: 433-444Crossref PubMed Scopus (137) Google Scholar, 5Dyda F. Klein D.C. Hickman A.B. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 81-103Crossref PubMed Scopus (383) Google Scholar). Previous work has shown that AANAT follows an ordered Bi Bi ternary complex mechanism in its catalysis of acetyl transfer, with acetyl-CoA binding to the enzyme first, followed by the binding of serotonin or tryptamine (6De Angelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Alternative substrates and pH-rate studies on AANAT revealed that ionizability of two or more groups, a potential catalytic base and acid, may be important in the reaction mechanism (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Using the knowledge that both substrates bind to the enzyme simultaneously, a potent inhibitor (Ki = 50 nm) was developed that incorporates both acetyl-CoA and serotonin (Fig. 1 B) (8Khalil E. Cole P.A. J. Am. Chem. Soc. 1998; 120: 6195-6196Crossref Scopus (65) Google Scholar). Moreover, it was shown that AANAT itself can catalyze the formation of the bisubstrate analog via a newly discovered “alkyltransferase activity” (Fig. 1 B) (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar, 10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This activity, catalyzed in an apparently subtly altered form of the acetyltransferase active site (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar, 10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), allows for the delivery of a cell permeable “prodrug” form of the inhibitor. The bisubstrate analog inhibitor has been used in complex with AANAT to obtain high resolution crystal structures of the enzyme (11Hickman A.B. Namboodiri M.A.A. Klein D.C. Dyda F. Cell. 1999; 97: 361-369Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 12Wolf E., De Angelis J. Khalil E.M. Cole P.A. Burley S.K. J. Mol. Biol. 2002; 317: 215-224Crossref PubMed Scopus (50) Google Scholar). These crystal structures show possible residues that may play a role in catalysis (Fig. 2). Based on these structural studies and preliminary kinetic analysis, Tyr-168, His-120, and His-122 have been proposed to be catalytic residues (11Hickman A.B. Namboodiri M.A.A. Klein D.C. Dyda F. Cell. 1999; 97: 361-369Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this work, we examined the mechanistic function of these three residues in greater detail to attempt to identify more definitive roles for these residues in AANAT catalysis. Not only should this knowledge be useful in relating AANAT to other GNAT family members, it may also provide clues to the development of specific inhibitors of AANAT that might be used to treat mood and sleep disorders (13Arendt J. Melatonin and the Mammalian Pineal Gland. Chapman and Hall, Inc., New York1995Google Scholar). Tryptamine,N ω-methyltryptamine, racemic α-methyltryptamine, 5,5′-dithiobis(2-nitrobenzoic acid), glutathione-agarose, coenzyme A, MES, MOPS, EPPS, and CHES were acquired from Sigma. Acetyl-CoA was purchased from AmershamBiosciences. Sucrose was purchased from Bio-Rad.N-Bromoacetyltryptamine (Fig. 1 B, compound1) and the bisubstrate analog (compound 2) were synthesized as previously described (8Khalil E. Cole P.A. J. Am. Chem. Soc. 1998; 120: 6195-6196Crossref Scopus (65) Google Scholar). All AANAT mutants were prepared by PCR-based site-directed mutagenesis and verified by DNA sequencing. The mutants were expressed as GST fusion proteins and purified as described previously (6De Angelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), with several exceptions. Protein-expressing Escherichia coli strains were grown as previously described, except that inductions with isopropyl-β-d-thiogalactopyranoside were performed at 16 °C for 22 h to enhance production of soluble protein. The GST-mutant fusion proteins were purified over a glutathione-agarose column after lysis by French press. The column (swelled from 0.8 g of resin) was equilibrated with 100 ml of lysis buffer prior to addition of the cleared lysate. The column was subsequently washed with 50 ml of lysis buffer and 200 mm NaCl, and the mutant proteins were eluted with 50 ml of lysis buffer, 110 mmNaCl, and 50 mm glutathione (pH 7.0) and collected in 10-ml fractions. Purity of the mutants (estimated to be >80% in all cases) was examined by 12% SDS-PAGE, and protein concentration was determined by Bradford analysis (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) referenced to bovine serum albumin. Mutant enzymes were studied as GST fusion proteins for the kinetic assays described. Previous work on the wild-type enzyme (6De Angelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and current studies on Y168F (data not shown) show that GST does not greatly alter the kinetic characteristics of acetyl- or alkyltransferase reactions of the enzyme. Values for both kcat and Km of GST fusion proteins were within 2-fold of enzymes assayed without the affinity tag. The acetyltransferase activity of the GST fusion mutants was measured using a non-continuous spectrophotometric assay involving the liberation of a colored product from the reaction of CoASH with 5,5′-dithiobis(2-nitrobenzoic acid) that was described previously (6De Angelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Assays were performed in a final volume of 0.3 ml in buffer consisting of 0.05 m sodium phosphate (pH 6.8), 0.5 mNaCl, 2 mm EDTA, and 0.05 mg/ml bovine serum albumin at 30 °C. Varying amounts of acetyl-CoA (0.025–10 mm) and tryptamine (0.2–30 mm) were added to the reaction mixture, and reactions were initiated with enzyme diluted in the buffer described above, with final enzyme concentrations ranging from 0.05 to 1 μm, depending upon the mutant. Reactions were quenched with a guanidinium solution (0.3 ml; 3.2 m guanidinium chloride in 0.1 m sodium phosphate (pH 6.8)), and 0.05 ml of 5,5′-dithiobis(2-nitrobenzoic acid) (2 mm) was added prior to taking readings at 412 nm. Reaction rates were measured under initial conditions (linear with respect to time and enzyme concentration, <10% turnover of the limiting substrate). Assays involving Km measurements were conducted with a near-saturating concentration (Km > 3) of the unvaried substrate. All viscosity assays were performed in the same buffer and under the same conditions as described above. The sucrose solutions and relative viscosities were the same as described (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Tryptamine Km values were assayed at each of the varied sucrose solutions, with the exception of H120A and H122A, for which the Km values were measured at the high and low points only and were not significantly altered in response to these changing sucrose levels. Viscosity studies were run with tryptamine on the Y168F, H120Q, H122Q, H120Q/H122Q, H120A, and H122A AANAT mutants with final enzyme concentrations of 500, 50, 50, 250, 100, and 100 nm, respectively. Controls were run for Y168F AANAT with α-methyltryptamine using a final enzyme concentration of 2 μm and for the H120Q and H122Q AANAT mutants with N ω-methyltryptamine at a final enzyme concentration of 250 nm. Viscosity effects on the steady-state kinetic parameters of the mutants were determined, as in Ref. 7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, from Equation 1, (kcat(control)/kcat(viscogen) versus relative viscosity)slope=k2/(k2+k30)Equation 1 where k2 is the rate constant for the chemical step and k3 0 represents the rate constant for product release in the absence of viscogen, as in previous studies (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). This equation assumes the formation of a fully saturated ternary complex, followed by the chemical reaction and then product release. Measurements of kcat and tryptamine Km were performed as described above and, under the conditions previously reported (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), over a pH range of 6.0–8.5. The buffers used in these reactions were as follows: MES (pH 6.0–6.7), MOPS (pH 6.7–7.6), EPPS (pH 7.7–8.5), and CHES (pH 8.3–8.5). All mutant enzymes had linear activities at each pH within the time used for the assays, and all were found to exhibit Michaelis-Menten kinetics at these pH values. Curve fits for kcat and kcat/Km data were calculated as described previously (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These were carried out with a range of inhibitor (compound 2) concentrations as described (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar) using a Dixon analysis to give an apparent Ki assuming a competitive inhibition model. Assays for alkyltransferase activity were carried out as described previously (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar, 10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly, the reaction conditions contained 50 mmsodium phosphate (pH 6.8), 0.05 mg/ml bovine serum albumin, 2 mm EDTA, and 500 mm NaCl at 30 °C. The products were separated and quantified by reversed-phase high pressure liquid chromatography and UV detection using the authentic materials as standards (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar, 10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The Km for N-bromoacetyltryptamine (compound 1) was determined using a range of 0.025–2.5 mm in the presence of a constant and near-saturating concentration of CoASH (1 mm). The measurement of the Km for CoASH employed a concentration range of 0.025–3.0 mm with fixed and saturating N-bromoacetyltryptamine (compound1; 2 mm). The final enzyme concentrations ranged from 3.5 to 20 μm; and in all cases, the limiting substrate was at least 5-fold higher than the enzyme concentration. In all cases, the background (nonenzymatic) rates were subtracted from the rate in the presence of enzyme to give the net enzymatic rate. Reaction rates were measured under initial conditions (linear with respect to time and enzyme concentration, <10% turnover of the limiting substrate). GST-AANAT Y168F protein was proteolytically cleaved of GST and purified over glutathione- and CoA-agarose resins as described previously (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar). Crystals of the Y168F AANAT mutant bound to compound 2 were obtained using previously published procedures (12Wolf E., De Angelis J. Khalil E.M. Cole P.A. Burley S.K. J. Mol. Biol. 2002; 317: 215-224Crossref PubMed Scopus (50) Google Scholar): space group C2221; unit cell dimensions a = 53,b = 69, and c = 89 Å; and one enzyme-inhibitor complex/asymmetric unit. Cocrystals were cryoprotected with 100 mm MES (pH 6.5), 30% polyethylene glycol 2000, 100 mm Mg(OAc)2, 2% (v/v) 2,4-methylpentanediol, 20% glycerol, 30 mm dithiothreitol, 20 mm spermidine, and 100 mm LiCl and frozen by immersion in liquid propane. Diffraction data were collected under standard cryogenic conditions on Beamline X4A (National Synchrotron Light Source, Brookhaven National Laboratory). Following data processing with DENZO/SCALEPACK (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), initial phases were obtained with our structure of the wild-type enzyme bound to compound 2(Protein Data Bank code 1L0C). Rigid body refinement and Powell minimization with CNS (15Brunger A. Adams P.D. Clore G.M. Gros P. Grosse-Kuntsleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) yielded clear evidence for the absence of the Tyr hydroxyl group at position 168 in an (Fo − Fc) difference Fourier synthesis (data not shown). The current 2.3-Å refinement model for the structure contains residues 30–195, compound 2 in one conformation, and 90 water molecules, giving an R-factor of 19.3% and a free R-value of 24.6% with excellent stereochemistry (16Laskowski R.J. MacArthur M.W. Moss D.S. Thonton J.M. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar) (overall G-value = 0.3; see Table IIIfor data collection and refinement statistics).Table IIIStatistics from the crystallographic analysis of Y168F bound to compound 2Intensity data processing Resolution20 to 2.3A Rsym3.7% (9.2%)3-aStatistics in parentheses denote high resolution shell data. No. of measurements75,910 No. of unique reflections7188 Completeness91.9% (93.0%)3-aStatistics in parentheses denote high resolution shell data. <I/ς(I)25.2 (14.1)3-aStatistics in parentheses denote high resolution shell data.Refinement statistics Resolution20 to 2.3 Å Completeness91.9% Rcryst/Rfree19.3/24.6% <B> for Y168F AANAT/222/33Å2 r.m.s.d. bond length0.007 Å r.m.s.d. bond angle1.3 ° Cross-validated Luzzati coordinate error3-bThe cross-validated Luzzati and sigmaa coordinate errors for wild-type AANAT are 0.40 and 0.34 Å, respectively.0.34 Å Cross-validated sigmaa coordinate error3-bThe cross-validated Luzzati and sigmaa coordinate errors for wild-type AANAT are 0.40 and 0.34 Å, respectively.0.31 Å3-a Statistics in parentheses denote high resolution shell data.3-b The cross-validated Luzzati and sigmaa coordinate errors for wild-type AANAT are 0.40 and 0.34 Å, respectively. Open table in a new tab Based on the x-ray structure of AANAT bound to compound 2, Tyr-168 appears to make a hydrogen bond with the CoA sulfur atom (11Hickman A.B. Namboodiri M.A.A. Klein D.C. Dyda F. Cell. 1999; 97: 361-369Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 12Wolf E., De Angelis J. Khalil E.M. Cole P.A. Burley S.K. J. Mol. Biol. 2002; 317: 215-224Crossref PubMed Scopus (50) Google Scholar). Tyr-168 was mutated to phenylalanine by site-directed mutagenesis to prevent the side chain from being able to participate in conventional hydrogen bonding or proton donation. This Y168F AANAT protein was expressed and purified in a manner similar to the wild-type enzyme. Interestingly, attempts at expressing Y168A AANAT were unsuccessful, presumably due to the instability of this protein. However, the Y168F AANAT mutant displayed catalytic activity that was linear with time and followed Michaelis-Menten kinetics, suggesting that it was sufficiently stable for kinetic studies. Kinetic analysis of Y168F AANAT showed a 30-fold lower kcatcompared with wild-type AANAT and altered Km values for both substrates (Table I). The Km value for acetyl-CoA decreased to 0.03 mm, whereas the tryptamine Km rose to 2.3 mm.Table ISteady-state kinetic values for AANAT and AANAT mutants in acetyltransferaseAANAT formkcatAcetyl-CoA KmTryptamine Km(s−1)(mm)(mm)Wild-type1-aTaken from Ref. 6.25 ± 10.29 ± 0.0130.17 ± 0.014Y168F0.75 ± 0.040.03 ± 0.022.3 ± 0.32H120Q24.5 ± 0.700.67 ± 0.031.2 ± 0.11H122Q18.1 ± 1.110.791 ± 0.084.78 ± 0.62H120Q/H122Q3.1 ± 0.071.11 ± 0.1410.9 ± 0.63H120A5.6 ± 0.151.3 ± 0.117.8 ± 0.59H122A8.9 ± 0.080.86 ± 0.062.8 ± 0.09H120A/H122A0.094 ± 0.0037.6 ± 0.604.6 ± 0.40H120E0.041-bApproximate due to the instability of the enzyme.H122E2.1 ± 0.031.5 ± 0.203.0 ± 0.15Values were measured as described under “Experimental Procedures.”1-a Taken from Ref. 6De Angelis J. Gastel J. Klein D.C. Cole P.A. J. Biol. Chem. 1998; 273: 3045-3050Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar.1-b Approximate due to the instability of the enzyme. Open table in a new tab Values were measured as described under “Experimental Procedures.” To further understand the significance of these changes, analysis of the effects of the microviscogen sucrose on the kinetic parameters was performed. With appropriate controls, measurement of viscosity effects can be useful in determining the rate-limiting step of enzyme reactions because they can affect the diffusional processes, but not the chemical step(s) (17Blacklow S.C. Raines R.T. Lim W.A. Zamore P.D. Knowles J.R. Biochemistry. 1988; 27: 1158-1167Crossref PubMed Scopus (248) Google Scholar). Previous viscosity studies (kcat(control)/kcat(viscogen) versus relative viscosity) have shown that the rate-limiting step of AANAT when tryptamine is used as a substrate is predominantly a diffusion-controlled process (slope = +0.75, theoretical maximum = 1.0), most likely product (CoASH) release (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In contrast, the chemical step of the reaction is rate-limiting when a “poor substrate” such as N ω-methyltryptamine is substituted for tryptamine (slope = +0.10) (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Poor substrates are often used as a control to detect any nonspecific effects of the viscogen on the enzyme (18Cole P.A. Burn P. Takacs B. Walsh C.T. J. Biol. Chem. 1994; 269: 30880-30887Abstract Full Text PDF PubMed Google Scholar). No detectable activity was seen with Y168F AANAT and N ω-methyltryptamine as a substrate. However, we showed that α-methyltryptamine behaves as a poor substrate with Y168F AANAT, with kcat = 0.013 s−1and Km = 4.2 mm, whereas with wild-type AANAT, α-methyltryptamine shows a kcat similar to the tryptamine reaction (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The slope of the kcat(control)/kcat(viscogen) versus relative viscosity plot for Y168F was found to be +0.38 using tryptamine as a substrate and +0.03 with α-methyltryptamine (Fig. 3). As expected with a poor substrate, the slope was virtually zero, indicating that the chemical step is indeed rate-limiting in this case. Despite the 30-fold lower kcat compared with the wild-type enzyme, the non-zero viscosity effect seen with tryptamine suggests unexpectedly that the chemical step is not fully rate-limiting in acetyl transfer catalyzed by this mutant. Thus, both the product release step and the chemical step are slowed by this mutation. Given the observation that the Km for acetyl-CoA is 10-fold lower with this mutant and the data described above, these results suggest that CoASH has a higher affinity for Y168F and is more slowly released. The removal of an ionizable amino acid residue important in catalysis could result in a change in the pH-rate profile of an enzyme. The profile of log(kcat/Km)versus pH for wild-type AANAT from previous work (7Khalil E.M., De Angelis J. Cole P.A. J. Biol. Chem. 1998; 273: 30321-30327Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) shows that there is an acidic limb reflecting one ionizable group with a p Ka of 7.3 and a basic limb reflecting one ionizable group with a p Ka of 8.5 (Fig. 4 A). The pH-rate analysis of Y168F AANAT revealed a distinct change in the higher pH values of the pH-rate profile found on the log(kcat/Km)versus pH plot (Fig. 4 B). Although the acidic limb with a p Ka of 7.3 was maintained, the basic limb of the wild-type profile was now absent, suggesting that Tyr-168 has a p Ka of 8.5 and that Tyr-168 should be neutral for optimal activity. To further probe the active site structure of Y168F AANAT, the susceptibility of Y168F AANAT acetyltransferase activity to inhibition by the bisubstrate analog inhibitor (compound 2) was examined. Using a Dixon analysis, the extrapolated Ki of 50 nm was found to be essentially identical to that of compound 2 inhibition of the wild-type AANAT enzyme, arguing for the absence of large structural perturbation. The steady-state kinetic parameters for the alkyltransferase activity (9Khalil E.M., De Angelis J. Ishii M. Cole P.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12418-12423Crossref PubMed Scopus (46) Google Scholar, 10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Wolf E., De Angelis J. Khalil E.M. Cole P.A. Burley S.K. J. Mol. Biol. 2002; 317: 215-224Crossref PubMed Scopus (50) Google Scholar) of Y168F AANAT with N-bromoacetyltryptamine (compound 1) were measured and are displayed in Table II. They reveal a 290-fold reduction in kcat for the Y168F mutant enzyme compared with wild-type AANAT. The Km values for the substrates of this reaction indicate that the apparent affinity of both substrates for the enzyme increased. The Km for CoASH dropped 5-fold (comparable to the Km drop for acetyl-CoA in the acetyltransferase reaction), whereas that for N-bromoacetyltryptamine (compound 1) was reduced more dramatically by 120-fold. Because the chemical step is likely to be rate-determining for this mutant (10Zheng W. Scheibner K.A. Ho A.K. Cole P.A. Chem. Biol. 2001; 8: 379-389Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), we can make the assumption that rapid equilibrium conditions apply. Under these conditions, the measured substrate Km is representative of the dissociation constant (Kd). The fact that both the wild-type enzyme and mutant yielded similar Ki values for the bisubstrate analog (compound 2) indicates that the rate of product release for both enzymes is similar. Therefore, the reduced Km for N-bromoacetyltryptamine (compound 1) is suggestive of an" @default.
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