Electronic spectrum of the coenzyme chromophore

Spectroscopic studies have shown that the chromophoric system of the coenzyme absorbs at 390 nm, in a region clearly separated from the 280 nm peak of the protein moiety. This feature has been exploited for a variety of structural and kinetic studies 

96) and concluded the pA of the 3-hydroxy function to be 5.1 and the pyridinium nitrogen to be 8.6 (Fig. 40). This information allows one to interpret the pA, values of 3.1 and 8.3 obtained by Morozov et al. [92] for pyridoxal, as those corresponding to the 3-hydroxy and N-1 groups respectively (Fig. 41). It was noted [92] that the presence of a 5 -phosphate group on pyridoxal had practically no effect on the shape, half-width, or position of the absorption bands and hence pA s as noted above. 

In close analogy to 3-hydroxypyridine, the chromophores in pyridoxamine phosphate and other related species of types 2 and 3 (Fig. 42) possessing a tetrsihedral centre at C-4, absorb at about 330 nm. The presence of an aldehyde function at this position, as in pyridoxal phosphate (Fig. 42, 1) shifts the absorption spectrum to 390 nm. The conversion of the aldehyde into a Schiff base is attended by a slight hypsochromic shift, but the protonation of the Schiff base produces a dramatic bathochromic shift and species of type 5 (Fig. 42) absorb at approximately 420-440 nm. Guided by the data from chemical model systems we examine the spectroscopic information obtained in the formation of binary and ternary complexes with several 

The binary complex can exist as an equilibrium mixture of a number of species as shown in Fig. 43. The major species apparent in a number of binary complexes, e.g. glutamate.decarboxylase and aspartate aminotransferase [31,90] appear to be those absorbing at 420 and 333 nm which are attributed to structures 2 and 3 (Fig. 43) respectively as expected the ratio of these species is pH dependent. On the other hand, the ternary complex may exist as an equilibrium mixture composed of species from both binary and ternary complexes, thus producing a composite electronic absorption profile however, the ternary complex of aspartate aminotransferase exhibits only two major absorption maxima at 430 and 340 nm due to substrate-coenzyme Schiff base and enzyme bound pyridoxamine-P respectively [90]. It is interesting to note the spectra observed for aspartate aminotransferase 

Depending upon the relative rates of the reactions participating in the interconversion of various species in the ternary complex, one may expect to see spectropho-tometrically the presence of the quinonoid intermediate of type 6 (Fig. 42) in a number of pyridoxal-P-dependent enzymic reactions such an intermediate would be expected to possess a bathochromically shifted long wavelength absorption maximum. This has indeed been clearly viewed for the ternary complex of serine hydroxymethyltransferase with glycine as shown in Fig. 46, the absorption at 495 nm being attributed to the quinonoid intermediate [94]. 

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