Catalysis by Acetylcholinesterase
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 3834-38, October 1975 Biochemistry Catalysis by acetylcholinesterase: Evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis (enzyme mechanism/diffusion control/induced-fit conformational change/pH dependence/deuterium oxide isotope effects) TERRONE L. ROSENBERRY Departments of Biochemistry and Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 Communicated by David Nachmansohn, June 9,1975 ABSTRACT Inferences about the catalytic mechanism of The proposed intermediates include the initial Michaelis acetylcholinesterase (acetyicholine hydrolase, EC 3.1.1.7) are complex E-RX and the acyl enzyme ER, for which evidence frequently made on the basis of a presumed analogy with has long been obtained (5, 6, 1). The pH dependence of sub- chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic strate hydrolysis for chymotrypsin and other serine hydro- properties of the two have been observed. In this report par- lases suggests general acid-base catalysis by a group in the ticujar attention is focused on the second-order reaction con- free enzyme with a pKai of 6 to 7. Furthermore, Hammett stant, kcat/KapD While the reported pH dependence and deu- relationships with positive rho values are found with chymo- terium oxide isotope effect associated with this parameter trypsin both for deacylation (7) and acylation (8) reactions for chymotrypsin are generally consistent with simple mod- and indicate rate-limiting general base catalysis. Deacyla- els involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholi- tion rates are typically reduced in deuterium oxide by fac- nesterase. The apparent pKa of kcat/Kapp for acetylcholinest- tors of 2 to 3 (9), in agreement with this indication. Partial erase varies between 5.5 and 6.3 for neutral substrates and protonation generally accompanies loss of the leaving group involves nonlinear inhibition by [H+J. Deuterium oxide iso- during acylation (10). Crystallographic analysis of acyl chy- tope effects for kcat/Kapp range from 1.1 for acetylcholine to motrypsins has suggested that His-57 acts successively dur- 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate ing acylation, first as a general base for the attack of Ser-195 appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account on the carbonyl carbon and then as a general acid to assist for apparent pKa values and low deuterium oxide isotope ef- loss of the leaving group (11). Because of the symmetry of fects associated with low concentrations of phenyl acetate the proposed action of His-57, deacylation'is presumed to and isoamyl acetate. occur by a similar process. In the absence of the three-dimensional structure for Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) is acetylcholinesterase, inferences about its mechanism have classified as a serine hydrolase along with other esterases and been based both on studies of its substrate catalysis and on peptidases which show essentially irreversible active-site analogies drawn from chymotrypsin (see refs. 1, 12, and 13). phosphorylation (see ref. 1). Equivalent-weight determina- Yet aside from the similarities which classify it as a serine tions and peptide analysis indicate that phosphorylation oc- hydrolase, acetylcholinesterase differs significantly from curs only at a single serine residue, and the amino-acid se- chymotrypsin both in size and in catalytic properties. 11S quence about this residue shows significant homology acetylcholinesterase is a tetramer of essentially identical cat- among the enzymes in this class (1, 2). Three-dimensional alytic subunits, each with a molecular weight of about structures of several serine hydrolases have been determined 75,000 (14).' The active site includes an anionic group which by x-ray crystallography, and further striking structural aids in the binding of cationic substrates (see refs. 1, 12, and similarities have thus been revealed (3). The native polypep- 13). It is also an esterase as opposed to a pepfidase; acetyl- tide conformations of chymotrypsin (EC 3.4.21.1), trypsin, acetylcholinesterase is hydrolyzed some 106 times faster than and elastase are nearly superimposable. Blow et al. (4) have acetylchymotrypsin (ref. 15, and see Table 1). Although chy- reported that a dominant feature of these structures is a motrypsin is rapidly acylated 'by specific ester substrates "charge-relay" system of hydrogen bonds formed by the ac- (e.g., ref. 16), its catalytic machinery appears to have tive site serine hydroxyl, a histidine imidazole side chain, evolved for the particular stabilization of the transition state and a carboxylate side chain in linear array. The evolution- for acylation with specific amides, especially those with ary importance of this charge relay is demonstrated by its amino-acid amide leaving groups (17). identification in subtilisin and other serine hydrolases To decide whether the mechanism in Scheme 1 can ade- structurally unrelated to chymotrypsin (see ref. 3). quately account for observations on acetylcholinesterase, The discovery of the charge-relay structure coincides with some properties of the experimental parameters derived previous inferences about the catalytic mechanism of serine from Scheme 1 have been examined in this paper. Under hydrolases from kinetic studies. A minimal catalytic mecha- steady-state conditions of substrate hydrolysis ([RX] >> Etot, nism involves the enzyme species in Scheme 1. where Etot is the enzyme normality), the kinetic parameters X HO0 kcat and Kapp are defined by Eq. 1, where v is velocity. E + RX E-RX ER - E + ROH k_, + + + V = kcatEtot/(l + Kapp/[RX]) 111 H H H K;,," || K..Il The pH dependence of kcat/Kapp (the second-order rate) EH + RX EH RX ERH and kcat (the first-order rate) have long been formulated for 3834 Downloaded by guest on October 4, 2021 Biochemistry: Rosenberry Proc. Nat. Acad. Sci. USA 72 (1975) 3835 Table 1. Apparent pKa values associated with acetic acid ester substrates of acetylcholinesterase oI 9 A *A 0 -log R (, ) 0 kcat/Kapp kcat 0a 0 5- O& A Substrate PKai pKa2 pKai pKa2" 1.0 -0 Acetylcholine 0.1 M NaCl 6.3 >10.5 6.5 >10.5 Phenyl acetate j 0.1 M NaCl 5.5 10.5 6.56 >10.5 50 60 70 80 90 l00 1 MNaCI 5.5 9.8 6.1 10.1 FIG. 1. The pH dependence of phenyl acetate hydrolysis by acetylcholinesterase. R values were calculated as outlined in Mate- Values were determined as outlined in Materials and Methods. rials and Methods for the following parameters: (0) kcat/Kapp in 0.1 M NaCl; (A) kcatlKapp in 1.0 M NaCl; () kcat in 0.1 M NaCl; (-) kcat in 1.0 M NaCl. Scheme 1 under the assumption that all reversibly linked species are equilibrated (18). If protonation of a single group inhibits enzyme acylation, as indicated in Scheme 1, the pH lease of protons for acetate at low pH and for phenol and p- dependence of kcat/Kapp gives the pKal of this group in the nitrophenol at high pH, both in H20 and D20. free enzyme; the apparent pKa of kcat is a weighted average The second order phosphorylation rate for p-nitrophenyl- of the pKai' and pKali values for this group in E-RX and ER diethylphosphate was determined by monitoring the simul- (see ref. 12). The pH dependence of chymotrypsin is quite taneous hydrolysis of either methyl acetate or p-nitrophenyl consistent with this formulation. A pKa. of 6.8 for kat/Kapp acetate. Conditions were defined such that the ratio of the is observed for virtually all chymotrypsin substrates (9, 19); a velocity of hydrolysis of the acetic acid ester (at a concentra- similar pKai' is obtained for most E-RX species and a pKai, tion far below its Kapp) to the free enzyme normality was ef- of 7.0-7.2 is seen with acetylchymotrypsin ER (9). In con- fectively constant during the phosphorylation reaction; this trast, apparent pKal values which vary between 5.5 and 6.3 permitted direct continuous determination of the phospho- have been reported by Krupka (20, 21) for partially purified rylation rate by a technique previously applied to carba- bovine erythrocyte acetylcholinesterase. A pKa,' of 5.2-5.5 moylating agents (23, 22; see ref. 13). for E-RX was inferred from all substrates for which acetyla- The effects of pH or high ionic strength on the kinetic pa- tion is rate-determining, while deacetylation-limited sub- rameters kcat/Kapp and kcat were assessed in terms of R. For strates indicated a pKali" of 6.3 for the acetyl enzyme ER. pH studies, R is defined as the ratio of the value of the kinet- Of particular interest in these acetylcholinesterase studies is ic parameter at a given pH to that at pH 8.5. For ionic the suggestion that protonation of two groups in the free en- strength variations, R is the ratio at a given ionic strength to zyme can affect activity (21). that at 0.1 M NaCl. Values of the apparent inhibition con- In this report a variability in the pKai of kcat/Kapp for sev- stants Ka, and Kai" were estimated visually from the initial eral substrates of highly purified eel IIS acetylcholinesterase slopes of plots of 1hR versus [H'] (22). Curvature in these is confirmed. These pKal values and observed deuterium plots precluded the computer analysis used previously (22). oxide effects provide new information about the acylation of acetylcholinesterase. RESULTS General Effect of pH on the Hydrolysis of Acetic Acid Esters. The pH dependence of phenyl acetate hydrolysis is MATERIALS AND METHODS shown in Fig.