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Aspartate aminotransferase from wheat germ: purification and kinetic properties.
No full textA convenient method for the purification of aspartate aminotransferase [L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1)] from wheat germ is described. An overall purification of 150 fold was achieved. On polyacrylamide gel electrophoresis at pH 8.9 the purified enzyme revealed two protein bands both provided with enzymatic activity. The holoenzyme is readily resolved on conversion to the aminic form and gel-filtration. The apoenzyme is reactivated by pyridoxal-5-phosphate. Kinetic data indicate that a Ping-Pong mechanism is operative similar to that found for the tyrosine aminotransferase by Litwack and Cleland (1968). Phosphate ion behaves as a competitive inhibitor towards the coenzyme. The relatively low affinity between coenzyme and apoenzyme from wheat germ allowed the determination of the dissociation constants for coenzymes (pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate) and of the inhibition constant for phosphate
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 AGO 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 -12.5 kJ/mol (-0.9,-1.9
and -3 kcal/mol) respectively; the AGO for the binding of the coenzyme phosphate group
was shown to be more negative than -20.5kJ/mol (-4.9 kcal/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 intrinsic AGO appears to be used for
some associated endoergonic process; (2) the AH0 and AS0 of binding appear to be very
sensitive indicators of the correct alignment of the substrate-coenzyme analogues at the
active site
Coenzyme removal from pyridoxal-phosphate dependent enzymes by means of hydrazide of carboxymethyl-cellulose.
No full text
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