Stereochemistry of enzymic transamination

Autor: Dunathan Hc, Lawrence C. Davis, Kaplan M, Kury Pg
Rok vydání: 1968
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Zdroj: Biochemistry. 7:4532-4537
ISSN: 1520-4995
0006-2960
DOI: 10.1021/bi00852a049
Popis: An approach to the determination of the complete stereochemistry of enzymatic transamination is described. Stereospecificity in the enzymatic labilization of one of the 4-methylene protons of pyridoxamine has been demonstrated in the transamination of pyridoxamine catalyzed by apoglutamate-oxaloacetate transaminase. Both enantiomers of the 4-(CHD-NH2) pyridoxamine have been prepared. These compounds show T he large family of enzymes utilizing pyridoxal phosphate as cofactor catalyze a great variety of transformations of amino acids (Braunstein, 1963). In all cases the mode of action of the cofactor can be understood in terms of the original mechanism of Braunstein and Schemyakin (1953) and Snell (Metzler et ul., 1954)’ In this formulation all of the enzymatic reactions involve a common intermediate, the cofactor amino acid Schiff base. The properties of reaction, substrate, and stereospecificity are then imposed on this intermediate by the apoenzyme. Given the relative simplicity of this Schiff base intermediate and the limited numbers of conformations it can assume, one may hope to achieve a real understanding of the basis for reaction and stereospecificity in this group of enzymes. In an earlier paper we suggested that reaction specificity in pyridoxal phosphate enzymes must involve enzymatic control of the amino acid C,-N bond conformation (Dunathan, 1966). In this paper we begin to define the precise stereochemistry of enzymatic transamination. The fundamental step of transamination is the tautomerism or 1,3-prototropic shift shown in Figure 1. This simple reaction must take place within the confines of only a few stereochemical variables. These can be listed ~~ __ * From the Department of Chemistry, Haverford College, Haverford, Pennsylvania 19041. Receioed August 14 1968. This research was supported by U. S. Public Health Service Grant AM 09309 from the National Institute of Arthritis and Metabolic Diseases and by National Science Foundation Grant GY 4421. A preliminary account of this work was presented at the Conference on Chemical and Biological Aspects of Pyridoxal Catalysis, Moscow, Sept 1966. 1 The requirement for pyridoxal phosphate in phosphorylase (Fisher er al., 1958) cannot be explained by the Snell-Braunstein 4532 mechanism. the expected kinetic isotope effects in the enzymatic transamination. This effect provides a convenient way to compare the symmetries of monodeuteriopyridoxamine samples derived from different enzymes. It is suggested that the symmetry of the hydrogen labilized at the pyridoxamine 4-methylene group may be related to the symmetry of the amino acid substrate. A tentative assignment of the absolute symmetry of the monodeuteriopyridoxamines is made. simply: (1) the configuration at the amino acid a-carbon (C,); (2) the configuration of the proton added to the pyridoxal carbon (Cp); (3) the conformation about the C,-N single bond; (4) the conformation of the C,,= N double bond; and (5) the stereochemistry of the proton transfer (see Figure 1). In any real enzymatic transamination, the configuration of the amino acid is known, the conformation about the C,=N is almost certainly “trans,” and the C,-N conformation is restricted to one of the two in which the C,-H bond lies in a plane perpendicular to the plane of the cofactor B system (Dunathan, 1966). The only real unknowns are the configuration of the proton added to the pyridoxal carbon, the choice of the C,-N conformation, and the stereochemistry of proton transfer. This transfer will be called cis if the CH bond breaking and making both take place on the same side of the T system plane or trans if on opposite sides. These five variables have an “algebraic” relationship to each other such that knowledge of any four of the five will define the fifth. Definition of all five will in turn define a large part of the geometry of the active site and will restrict considerably the mechanistic possibilities for the prototropic shift. In stating these unknowns we have assumed enzymatic stereospecificity in adding and removing a proton at the pyridoxamine 4-methylene carbon. This is certainly to be expected considering the number of examples of enzymatic discrimination between the protons of a XCH2-Y grouping (Rose, 1966). In this paper we describe proof of this stereospecificity at pyridoxamine and the isolation of the two monodeuteriopyridoxamine enantiomers. We have made tentative assignment of the absolute configuration of these compounds. D U N A T H A N , D A V I S , K U R Y , A N D K A P L A N In these experiments we have chosen to work with the deuterium-labeled cofactor since this holds the possibility of the involvement of optical rotatory dispersion-circular dichroism and nuclear magnetic resonance measurements in determining the relative and absolute symmetries of monodeuteriopyridoxamine. Since these determinations require considerable quantities of the cofactor molecules, it was essential to use an enzyme which transaminates but does not tightly bind the cofactor. Several enzymes are known which transaminate the nonphosphorylated forms of the BG cofactors (Wada and Snell, 1962a,b). In these reactions the pyridoxal and pyridoxamine act as freely diffusable substrate molecules. We have used the apoenzyme form of glutamate-oxaloacetate transaminase which has been shown to catalyze reaction I (Wada and Snell, 1962a). pyridoxamine + a-ketoglutarate E pyridoxa; +
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