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amino acid portions of the P- Pxy-Tyr analogs. Also, the aldehyde group of pyridoxal-P displayed a special property, markedly increasing the binding of coenzyme to these antibodies but not to antibodies against the coenzyme alone. References Bartlett, G. R . (1959), J . Biol. Chem. 234, 466. Cordoba, F., Gonzalez, C., and Rivera, P. (1 966), Biochim. Biophys. Acta 127, 15 1. Cordoba, F., Rivera, P., Calderon, J., and Agundes, C. (1 970), Immunochemistry 7 , 543. Dempsey, W. B., and Christensen, H. N. (1962), J . Bioi. Chem. 237, 11 3. Eisen, H. M. (1964), in Methods in Medical Research, Eisen, H. M., Ed., Chicago, Ill., Year Book Medical Publishers, Inc., pp 106, 115. Freedman, M. H., Grossberg, A. L., and Pressman, D. (1968a), J . Biol. Chem. 243, 6186. Freedman, M . H., Grossberg, A. L., and Pressman, D. ( 1 968 b), Biochemistry 7, 194 1. Gornall, A. G., Bardwill, C. S., and David, M. M . (1949), J . Biol. Chein. 177, 75 1.
Itzhaki, R. F., and Gill, D. M. (1964), Anal. Biochem. 9, 401. Karush, F. (1956), J. Amer. Chem. SOC.78, 5519. Kitagawa, M., Grossberg, A. L., Yagi, Y., and Pressman, D. (1 967), Immunochemistry 4 , 197. Lang, C. A. (1958), Anal. Chem. 30, 1692. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951), J . Biol. Chem. 193, 265. Pauling, L., Pressman, D., and Grossberg, A. L. (1944), J . Amer. Chem. SOC.66, 784. Peterson, E. A., and Sober, H. A. (1954), J . Amer. Chem. SOC.76, 169. Raso, V., and Stollar, B. D. (1973), J . Amer. Chem. SOC. 95, 1621. Rodwell, V. W., Volcani, B. E., Ikawa, M., and Snell, E. E. (1958), J . Biol. Chem. 233, 1548. Rubenstein, W . A., and Little, J. R. (1970), Biochemistry 9, 2106. Scatchard, G. (1949), Ann. N . Y. Acad. Sci. 51, 660. Stelos, P. ( 1 967) in Handbook of Experimental Immunology, Weir, D. M., Ed., Philadelphia, Pa., F. A. Davis Comp., p 3.
The Antibody-Enzyme Analogy. Comparison of Enzymes and Antibodies Specific for Phosphopyridoxyltyrosine+ Vic Rasot and B. D. Stellar*
ABSTRACT: Reduced Schiff base compounds of pyridoxal-P and tyrosine, which were used to induce specific antibodies described in the preceding article (V. Raso and B. D. Stollar, Biochemistry, 1975), caused active site-directed inhibition of tyrosine transaminase and tyrosine decarboxylase. The antibodies, studied as analogs of enzymes, were able to bind an unsaturated Schiff base catalytic intermediate, as shown by equilibrium dialysis and absorbance difference
spectroscopy. Schiff base formation can proceed while the pyridoxal-P and tyrosine are within the antibody combining site, but the rate of this bimolecular condensation within the sites was not greater than the rate in free solution. Antibody did effect a small rate enhancement for the pyridoxalP-catalyzed transamination of L-tyrosine. These results are discussed in light of current ideas in the mechanisms of enzyme catalysis.
T h e requirement for a good fit between protein and ligand in the formation of an enzyme-substrate complex provides adequate explanation for the specificity displayed in enzyme-catalyzed reactions. The extraordinarily high reaction velocities produced by enzymes are more difficult to account for and the problem of describing the mechanisms of these rate accelerations has remained an enigma for biochemists. Fundamental similarities between enzymes and antibodies are manifest a t the levels of binding specificity, strength of interaction, and kinetics of formation and dis-
ruption of ligand-protein complexes. It therefore appeared that an immunochemical approach to this problem might help to elucidate some aspects of the nature of enzyme catalysis. Following such an approach, we have obtained antibodies with combining site specificities similar to those of pyridoxal-P enzymes that act on the substrate tyrosine. A previous article presented the design, synthesis, and chemical and structural characterization of two compounds which resemble the Schiff base intermediate of pyridoxal phosphate dependent enzymes that act on the substrate tyrosine (Raso and Stollar, 1973). The preceding work (Raso and Stollar, 1975) described methods for attachment of these analogs to protein carriers and insoluble cellulose supports to provide both antigens for antibody induction and affinity adsorbants for isolation of the antibodies. The purified antihapten antibodies were characterized and compared with respect to their ligand binding properties. The present article describes the interaction of the haptens, as inhibitors, with two tyrosine-utilizing pyridoxa1-P-
+ From the Molecular Basis of Biological Phenomena Program and the Department of Biochemistry and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts 021 11. Received June 27, 1974. This investigation was supported by Grants G B 29628 and G B 37937 from the National Science Foundation. $ Present address: Children’s Cancer Research Foundation and the Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 021 15.
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dependent enzymes. In addition, antibodies to the stable PPxy-Tyrl hapten were examined for their ability to interact with a pyridoxal-P-tyrosine Schiff base, and the kinetics of the bimolecular condensation that yields the Schiff base were measured in the presence of antibody. In further experiments, the transamination of L-tyrosine catalyzed by pyridoxal-P free in solution was compared with the same reaction occurring in the presence of the antihapten antibodies. Materials and Methods Sources for many of the materials have been cited in the preceding article (Raso and Stollar, 1975). L-Tyrosine ( 1 I4C-labeled) was purchased from New England Nuclear and L-tyrosine (side chain 2,3-3H-labeled) from Amersham Searle. Tyrosine Decarboxylase (EC 4.1.1.25). Streptococcus faecalis, grown on a vitamin B6-deficient medium, was the source of tyrosine apodecarboxylase (Gunsalus and Smith, 1957). The enzyme preparation and assay mixture were as described by Maruyama and Coursin (1968). The crude apoenzyme alone had only 2.5% of the activity it showed in the presence of added coenzyme. The order of addition of reagents for assay was: buffer, enzyme, inhibitor, pyridoxalP,and a final delivery of L-tyrosine-1- I 4 C t o start the reaction. A filter paper soaked with hydroxide of hyamine was inserted into the top portion of the test tube (Klein and Sagers, 1966), the tube was sealed with parafilm, and the reaction was stopped with acid, and I4CO2 that had adsorbed to the filter paper was determined as described by Klein and Sagers (1 966). Tyrosine Aminotransferase (EC 2.6.1.5). R a t liver tyrosine aminotransferase was induced and purified through the ammonium sulfate fractionation step as described by Hayashi et al. (1967). The crude enzyme was resolved of coenzyme by dialysis against 0.1 M potassium phosphate-0.005 M tyrosine (pH 6) (Hayashi et al.. 1967) and the resulting preparation alone had only 10% of the activity seen upon the addition of excess coenzyme. Reaction conditions described by these authors as assay 111 were used for determination of activity. The order of addition of reagents was: buffer, a-ketoglutarate, tyrosine, inhibitor, enzyme, and pyridoxal-P to begin the 15-min incubation period. 3-,~-Acetyl-3-aminotyrosine. Ten millimoles (1.96 g) of 3-amino-~-tyrosineand 30 mmol (4.1 g) of sodium acetate 3 H 2 0 were added to about 50 ml of water and the p H was adjusted to 4.8 with 2 N HCI; 10 mmol (1.92 g) of watersoluble carbodiimide was then added with continuous stirring, and the p H was maintained with 2 N HCI. After 20 min the material was loaded on to a 98 X 3.7 cm column of XE-64 ion exchange resin. Elution with water was monitored by the absorbance a t 285 nm; one major peak (700 ml) was obtained. This material was flash evaporated to a volume of about 20 ml, and the resulting precipitate was redissolved by heating the mixture on a steam bath; when it cooled, crystals were obtained. The crystals were washed gently with cold water. Mother liquor and washings could be further concentrated to obtain more crystals. The com-
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Abbreviations used are: Tyr(NHAc), 3-!V-acetyl-3-aminotyrosine; P-Pxy-Tyr(NHz), N-(5-phosphopyridoxyl)-3’-amino-~-tyrosine; P-Pxd
