Abstract
Tryptophan synthase alpha 2 beta 2 complex containing [4'-3H]pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates. Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate. The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4'. On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced [3H]pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding. Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-[2,3-2H2]serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%). According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3.
Highlights
Tryptophan synthase a& complex containing [4’-3H] pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates
Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine,presumably resulting from reduction of the intermediate Schiffs base formed between pyridoxal phosphate and a-aminoacrylate
The a subunit catalyzes the aldolytic cleavage of indoleglycerol phosphate (Reaction b), whereas the P2 subunit binds pyridoxal phosphate as a Schiffs base with the €-amino group of a lysine residue and catalyzes the P-replacement of the hydroxyl group of serine with indole to form tryptophan (Reaction c):
Summary
Phan l n thePresenceof excess indole and tryptophansynthase.This i-rerlne contaminant was removed by enzymatic COnYerSlOn t oC - tryptophan which was absorbed on charcoal.Pure o - s e v ~ n e was relrolated .Indolepropanol Phosphate (IPP) was d g i f tf r c m Or. KarperKirschner,Biolentrum Barel. Racem ~ c[4'-3H]PM was prepared by t h e method O f Ounathan g 1.( 1 5 ). Alkalln ep hmphatase and aspartatetransaminase were obtained from Baehringer Hannheim The latter was converte dt ot h e apo e n z m as dercrlbed by Martinez - Carrion gtt. PLP was removed from the ezB2 ccmplexby amodificatio no ft h e method OfMiles and HDrlguchi ( 1 2 ) : n202 EOmPleX ( 2 0 rng/mL) i n 0.05 M sodium N.N-bir(2-hydraxyethyl)-glycine buffer( Buffer. Pyridoxinephosphate was synthesize ds imilarl yb yr eductionOf PLP inthe absence of amino a m d
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