Threonine synthase (TS), the last enzyme of the threonine biosynthetic pathway, catalyzes L-threonine formation from L-homoserine phosphate (HSerP; Km = 0.5 mM, V = 440 min-1) and DL-vinylglycine. Furthermore, TS catalyzes beta-elimination reactions with L-serine (Km = 150 mM, V = 4.7 min-1), DL-3-chloroalanine, L-threonine, and L-allo-threonine as substrates to yield pyruvate or alpha-ketobutyrate, while L-alanine, L-2-aminobutanoic acid, and L-2-amino-5-phosphonopentanoic acid are substrates for half-transamination reactions to form the pyridoxamine form of the enzyme and the corresponding alpha-keto acid. Spectral analyses of all these reactions revealed the transient formation of strongly absorbing long-wavelength chromophores (lambda max = 440-445 nm), implying the accumulation of the corresponding pyridoxaldimine p-quinonoidal intermediates. HSerP turnover was competitively inhibited by L-3-hydroxyhomoserine phosphate 1 (Ki = 0.050 mM), L-2,3-methanohomoserine phosphate 2 (Ki = 0.010 mM), L-2-amino-3-[(phosphonomethyl)thio)]propanoic acid 5 (Ki = 0.011 mM) and DL-E-2-amino-5-phosphono-4-pentenoic acid 10 (Ki = 0.54 mM). 5 and 10 induced the formation of long-wavelength quinonoidal chromophores (lambda max = 458 and 460 mm, epsilon 47,000 and 30,000 M-1 cm-1), while incubation with either 1 or 2 induced only minor spectral changes. DL-2-Amino-3-[(phosphonomethyl)amino)]propanoic acid inactivated TS (Ki = 0.057 mM, kinact = 1.44 min-1) with 1:1 stoichiometry, transient formation of a 450-nm chromophore, and finally bleaching of any absorbance at wavelengths longer than 320 nm. Z-2-Amino-5-phosphono-3-pentenoic acid 8 is the unusual amino acid found in the peptide antibiotics of the plumbemicin and rhizocticin families. Racemic 8 irreversibly inhibited TS (Ki = 0.1 mM, kinact = 1.50 min-1) with 1:1 stoichiometry and the concomitant formation of a 482-nm chromophore (epsilon approximately 30,000 M-1 cm-1). DL-E-2-Amino-5-phosphono-3-pentenoic acid was a less potent irreversible inhibitor of TS (Ki = 0.4 mM, kinact = 0.25 min-1), inducing absorption maxima at 462 and 500 nm. The acetylenic amino acid DL-2-amino-5-phosphono-4-pentynoic acid 12 bound to TS (KD = 0.38 mM) forming a quinonoidal chromophore (lambda max = 452 nm, epsilon approximately 30,000 M-1 cm-1), but inhibition of the enzyme by 12 could not be detected under assay conditions even at high inhibitor concentrations. Mechanisms consistent with these observations are proposed.

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Extracts of Synechococcus leopoliensis(Anacystis nidulans) contain two forms of D‐fructose‐1,6‐bisphosphatase(EC 3.1.3.11) previously designatedas forms A and B [Gerbling, K.‐P., Steup, M., and Latsko, E. (1984) Arch. Microbiol. 137, 109–114]. Form B, which probably represents the mahor part of the total extractable fructose‐1,6‐bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non‐denaturing polyacrylamide gel electrophoresis, and cross‐linking with bis(sulfosuccinimidyl) suberate revealed that the fructose‐1,6‐bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose‐1,6‐bisphosphate and Mg2+. The dimer–tetramer interconversion was readily reversible. The results provide evidence for a two‐step activation of fructose‐1,6‐bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate‐dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate‐dependent interconversion was detected with form A from S. leopoliensis.

ABSTRACITThe substrate specificity of purified fructose bisphosphatase form B from Synechococcus leopoliensis (EC 3.1.3.11; cf. K-P Gerbling, M Steup, E Latzko 1985 Eur J Biochem 147: 207-215) has been investigated. Of the phosphate esters tested only fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were hydrolyzed by the enzyme. Both sugar bisphosphates were cleaved at the carbon 1-ester. Fructose-and sedoheptulose bisphosphate stabilized the activated (i.e. tetrameric) state of the enzyme and prevented a slow inactivation that is observed in the absence of sugar bisphosphates. With the activated enzyme, kinetic constants (half-saturating substrate concentrations, maximal reaction velocity, and the catalytical constant) were similar for both fructose-and sedoheptulose bisphosphate. The data suggest that fructose bisphosphatase form B from Synechococcus leopoliensis can catalyze both bisphosphatase reactions within the reductive pentose phosphate cycle.In the reductive pentose phosphate cycle two sugar bisphosphate esters, FBP' and sedoheptulose 1,7-bisphosphate, are cleaved specifically at the carbon 1-ester. Both phosphatase reactions probably play an important role in regulating the flux through the entire cycle (12, 17; but see 7). However, conflicting data have been reported on the enzymology of both phosphatase activities. In some photo-and chemoautotrophic cells, fructose and sedoheptulose bisphosphatase activities have been attributed to distinct enzymes specific either for sedoheptulose bisphosphate or FBP (13,20). In contrast, in other organisms fructose bisphosphatase has been shown to hydrolyze sedoheptulose bisphosphate as well (1,2,18) and the existence of a sedoheptulose bisphosphate specific phosphatase has been questioned (1 1).

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