Human Plasma Lecithin:Cholesterol Acyltransferase (LCAT) On the role of essential carboxyl groups in catalysis
LCAT is one of the three major enzymes of plasma lipoprotein metabolism and catalyses the transacylation of the fatty acid at the sn- 2 position of lecithin to the 3-hydroxyl group of cholesterol forming lysolecithin and cholesteryl ester within the plasma compartment. In a series of reports (1-4) we elucidated the chemical catalytic mechanism of this important enzyme which now appears to bear some similarities to that of the recently discovered lecithin:retinol acyltransferase (LRAT) (5). Incorporating the previous observation that LCAT can esterify lysolecithin present in LDL (6) and the recent observations of Sorci Thomas et al. (7) the major features of the LCAT catalytic mechanism can be summarized as shown in Figure 1. The sn-2 carbonyl carbon of lecithin, present in plasma HDL, is initially subjected to nucleophilic attack by the oxygen atom of Serine-181 with the resultant formation of a transient tetrahedral adduct which decays with bond cleavage to yield a fatty acylated serine residue. The fatty acid is then internally transacylated to one of two vicinal cysteine residues (Cys-31 and Cys-184) by a mechanism that appears irreversible. Either cysteine residue can then donate its fatty acid moiety to cholesterol forming cholesteryl ester. The lysolecithin acyltransferase or LAT reaction is proposed to involve only Ser-181 which becomes acylated upon lecithin cleavage and donates its fatty acid back to another lysolecithin molecule when the concentration of the lysolipid is sufficiently high. Cholesteryl ester can be both formed and cleaved by LCAT (7). During cleavage the fatty acid is retained by the enzyme and transferred to another cholesterol molecule. Fatty acid originating as cholesteryl ester does not appear to be transferred to lysolecithin (7) and we thus propose that either of the vicinal cysteine residues mediates the cleavage of cholesteryl ester with the formation of a fatty acylated residue. This is designated as the CAT or cholesterol acyltransferase reaction in Fig. 1. The enzymatic cleavage of ester and amide bonds by serine esterases, proteases and triacylglycerol lipases is thought to involve a "catalytic triad" of three residues, serine, histidine and aspartic acid (8-10). These residues participate in a proton relay, the function of which is to retain the catalytic serine hydroxyl proton and thereby assist the nucleophilic attack of the serine oxygen atom upon the carbonyl carbon of the ester or amide bond.
Our previous studies utilizing specific chemical modification of highly purified LCAT (1) and the inhibitor, phenylboronic acid (2) had clearly demonstrated the involvement of a single serine and histidine residue in the first part of the LCAT reaction, lecithin cleavage. In an attempt to determine if a proton relay system involving aspartic acid is present within the catalytic site of LCAT we have recently evaluated the requirement for functional carboxyl groups in lecithin cleavage by LCAT. Our proposed mechanism for the first portion of the LCAT reaction is as shown in Fig. 2.
Our approach was to expose highly purified human plasma LCAT to 25 mM 1-Ethyl- 3-(3'-dimethylaminopropyl)-carbodiimide (EDAC), a specific carboxyl group reagent, in the presence of I-[14C] Glycine methyl ester which replaces the carbodiimide and radiolabels the activated carboxyl group. The results of these experiments are shown in Fig. 3 which clearly demonstrates a progressive, time dependent inactivation of LCAT by carbodiimide treatment with a concomitant increment in the number of carboxyl groups modified. Stoichiometric analyses of these data in the reciprocal plot shows that a maximum of three carboxyl groups are modified upon complete inactivation of the enzyme. Residues present within the catalytic site of an enzyme are normally afforded protection against chemical modification when incubated in the presence of their substrate(s). Contrary to the expected result, exposure of
LCAT to 50 mM EDAC in the presence of an artificial proteoliposome substrate containing Lecithin:Cholesterol:apo A-I (3.2:250:0.8 mol:mol:mol) completely failed to protect LCAT against inactivation.
However, increasing the cholesterol content of the proteoliposome relative to a fixed mass of lecithin and apo A-1 resulted in a progressive increment in the ability of these substrates to protect the enzyme against carbodiimide induced inactivation as shown in Fig.
4. Furthermore, proteoliposome with increased cholesterol content were better substrates for LCAT mediated cholesteryl ester formation than those with lower cholesterol content as shown in Table 1. Proteoliposomes with apo A-I content bteween 0.2 and 2.4 mol relative to 250 mol of lecithin and 12.5 mol of cholesterol were all equally ineffective in protecting LCAT against EDAC inactivation. HD , in contrast, effectively protected the enzyme. We therefore conclude that as the cholesterol to lecithin ratio of the artificial substrates approach that of HDL3, a natural substrate of LCAT, their catalytic efficiency as reflected by their ability to fit the catalytic site is increased. There is one further consideration with respect to this and our previous (1) substrate protection experiments in which we had shown that the proteoliposome containing the least amount of cholesterol (Lecithin:Cholesterol:apo A-1 (3.2:250:0.8 mol:mol:mol)) adequately protected the catalytic serine, histidine and cysteine residues against inactivation by chemical modification. In this instance it should be noted that the serine and cysteine residues would be fatty acylated during incubation with the substrate and thus unavailable to interact with the chemical modifiers. The histidine residue proximal to the catalytic serine residue may well be subject to steric hindrance following fatty acylation of the serine and thus protected against chemical modification. The carboxyl group of aspartic acid present within a proton relay system although "bmied" within the tertiary strucn1re of the protein is generally quite removed from the catalytic serine residue and does not participate directly in the catalytic mechanism via the formation of a transient tetrahedral adduct or via the formation of a relatively stable acylated species. Thus protection of the carboxyl groups of aspartic acid residues participating within a catalytic triad against chemical modification would
be more dependent upon enzyme substrate affinity and the extent to which the substrate conformed to the geometry of the catalytic site. This possibility appears to be supported by the data in Fig.4 and Table 1.
In conclusion, these data clearly show that a maximum of three functional carboxyl groups are required for the LCAT mediated formation of cholesteryl ester from lecithin and cholesterol. The requirement for functional carboxyl groups indicates, but does not prove,
that a Ser - His - Asp catalytic triad similar to that present in serine-histidine esterases, proteases and triacylglycerol lipases may be operative in human plasma LCAT.
REFERENCES
1) Jauhiainen,M., and Dolphin,P.J. (1986) J. Bioi. Chern. 261, 7032-7043.
2) Jauhiainen,M., Ridgway,N.D., and Dolphin,P.J. (1987) Biochim. Biophys. Acta. 918, 175-188.
3) Jauhiainen,M., Stevenson,K.J., and Dolphin,P.J. (1988) J. Bioi. Chern. 263, 6525- 6533.
4) Jauhiainen,M., Yuan,W., Gelb,M.H., and Dolphin,P.J. (1989) J. Bioi. Chern. 264, 1963-1967.
5) MacDonald,P.N., and Ong,D.E. (1988) J. Bioi. Chern. 263, 12478-12482.
6) Subbaiah,P.V., Albers,J.J., Chen,C-H., and Bagdade,J.D. (1980) J. Bioi. Chern. 255, 9275-9280.
7) Sorci-Thomas,M., Babiak,J., and Rudel,L.L. (1990) J. Bioi. Chern. 265, 2665-2670.
8) Kraut,J. (1977) Ann. Rev. Biochem. 46, 331-358.
9) Brady,L., Brzozowski,A.M., Derewenda,Z.S., Dodson,E., Dodson,G., Tolley,S., Turkenberg,J.P., Christiansen,L., Hugh-Jensen,B., Norskov,L., Thim,L., and Menge,U. (1990) Nature 343,767-770.
10) Winkler,F.K., D'Arcy,A., and Hunziker,W. (1990) Nature 343,771-774.
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