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The binding of the coenzyme pyridoxal 5'-phosphate and analogues of the substrate-coenzyme complex to tyrosine decarboxylase
No full textPhosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) -3.8. -8.4 and -12.5kJ/mol (-0.9, -1.9 and -3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than -20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic process; (2) the delta H0 and delta S0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme and analogues at the active site
Different reactivity of mitochondrial and cytoplasmic aspartate aminotransferases toward an affinity labeling reagent analog of the coenzyme.
No full textThe two isoenzymes of aspartate aminotransferase from pig heart have been reacted with a derivative of the coenzyme, 4'-N-(2,4-dinitro-5-fluorophenyl) pyridoxamine-5'-phosphate, which is a potential affinity labeling reagent. The derivative has a great affinity for both isoapoenzymes. In the cytosolic isoenzyme, the reversible binding is followed by a covalent labeling of the epsilon amino group of lysine 258, which usually forms an aldimine bond with pyridoxal-5'-phosphate. In the mitochondrial isoenzyme, no labeling occurs at the active site. The different reactivity indicates that a small but definite difference exists in the geometry of the two active sites. In the cytosolic isoenzyme also a sulfhydryl group outside the active site region, namely cysteine 45, reacts, but not by an affinity labeling mechanism. In both isoenzymes, the reversibly bound reagent slowly undergoes a splitting reaction by which pyridoxal-5'-phosphate is regenerated and activity re-established; the rate of this reaction is not fast enough to impaire the labeling potential of the reagent
[Separation of pyridoxal phosphate dependent enzymes by means of carboxymethylcellulose hydrazide]
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