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A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK · B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK · B was exclusively alkylated with iodoacetamide (R · CAM-UK · B) after reduction with dithiothreitol in 0.3 M guanidine · HCl in the presence of the competitive inhibitor N&alpha:-benzoyl-L-argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194-Cys222 whose SS loop contained the active-site Ser198, as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK · B into R · CAM-UK · B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK · B (pH 9.4) and R · CAM-UK · B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in k... in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or Nalpha;-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK · B was lowered to 28% or 35% for R · CAM-UK · B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194-Cys222 scission. [ABSTRACT FROM AUTHOR]

Incubation of wild-type ferredoxin (Fd) with Chlamydomonas reinhardii crude extract in the presence of a carboxyl activator resulted in the formation of a unique 1:1 covalent complex with nitrite reductase. However, glutamate synthase was able to form two covalent complexes of Fd: GOGAT with 1:1 and 2:1 stoichiometries. These complexes were functional only when reduced methyl viologen was used as electron donor. Kinetic studies of complex formation suggested the presence of two Fd-binding domains with similar affinity for Fd in the glutamate synthase molecule. Using site-directed mutagenesis with recombinant Fd. we have shown that Fd-Glu91 is directly involved in Fd interaction with glutamate synthase and nitrite reductase. Moreover, a negative core of residues in the α1 helix of Fd was also critical in binding the enzymes. These data highlight the analogy in the Fd-binding sites of nitrite reductase and glutamate synthase, which may compete for the electrons coming from the photosynthetic chain. [ABSTRACT FROM AUTHOR]