The catalytic mechanism of 1-aminocyclopropane-1-carboxylic acid oxidase.

It has been proposed that 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to ethylene via N-hydroxyl-ACC as an intermediate. However, due to its chemical instability the putative intermediate has never been isolated. Here, we have shown that a purified recombinant ACC oxidase can utilize alpha-aminoisobutyric acid (AIB), an analog of ACC, as an alternative substrate, converting AIB into CO2, acetone, and ammonia. We chemically synthesized the putative intermediate compound, N-hydroxyl-AIB (HAIB), and tested whether it serves as an intermediate in the oxidation of AIB. When [1-(14)C]AIB was incubated with ACC oxidase in the presence of excess unlabeled HAIB as a trap, no labeled HAIB was detected. By comparing the acetone production rates employing HAIB and AIB as substrates, the conversion of HAIB to acetone was found to be much slower than that of using AIB as substrate. Based on these observations, we conclude that ACC oxidase does not catalyze via the N-hydroxylation of its amino acid substrate. ACC oxidase also catalyzes the oxidation of other amino acids, with preference for the D-enantiomers, indicating a stereoselectivity of the enzyme.

Site-directed mutagenesis of lysine382, the activator-binding site, of ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Previous studies have shown that a highly conserved lysyl residue (Lys 419) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator [Charng, Y., Iglesias, A. A., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys382), suggesting that this residue might be also located within the activator-binding site [Charng, Y., Iglesias, AA., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Site-directed mutagenesis of Lys382 of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys382 with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P(i), and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate.

Mutagenesis of an amino acid residue in the activator-binding site of cyanobacterial ADP-glucose pyrophosphorylase causes alteration in activator specificity.

The specificity for activator of ADP-glucose pyrophosphorylase is closely related to the corresponding major carbon-assimilation pathway. The enzyme from Escherichia coli is mainly activated by fructose-1,6-P2, while the cyanobacterial, algal, and higher-plant enzymes are activated by 3-P-glycerate. Previous results have shown that Lys39 of the E. coli enzyme is involved in the binding of fructose-1,6-P2 while for the Anabaena enzyme, lysine residues 382 and 419 have been shown to be involved in the binding of 3-phosphoglycerate. This report shows that if Lys419 of the Anabaena enzyme is changed to glutamine, activation of the cyanobacterial enzyme by fructose-1,6-P2 becomes more effective than that of 3-P-glycerate at lower concentrations. Kinetic studies show that fructose-1,6-P2 competitively inhibits 3-P-glycerate activation of the Anabaena wild-type enzyme, suggesting that these two compounds bind to the same site. Thus a change of one amino acid at the activator binding domain can affect the specificity of activation of the Anabaena ADP-glucose pyrophosphorylase.

Structure-function relationships of cyanobacterial ADP-glucose pyrophosphorylase. Site-directed mutagenesis and chemical modification of the activator-binding sites of ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Chemical modification studies of spinach leaf ADP-glucose pyrophosphorylase with pyridoxal-P have shown that a highly conserved lysyl residue near the C terminus might be involved in the binding of 3-P-glycerate, the allosteric activator. Site-directed mutagenesis of the corresponding residue (Lys419) of the Anabaena enzyme was performed to determine the role of this conserved residue. Replacing Lys419 with either arginine, alanine, glutamine, or glutamic acid produced mutant enzymes with apparent affinities for 3-P-glycerate, 25-150-fold lower than that of the wild-type enzyme. The mutant enzymes, however, were still activated to a great extent at higher concentrations of activator suggesting that an additional site or residue was involved in the binding of the activator. These mutations caused lesser or no effect on the kinetic constants for the substrates and inhibitor, P(i), as well as on the catalytic efficiency and thermal stability. The results suggest that both the charge and size of lysine residue 419 are required for the proper binding of 3-P-glycerate. Chemical modification of the Anabaena wild-type enzyme with pyridoxal-P indicated that Lys419 was the only lysyl residue modified. We further performed the same experiment on the K419R mutant enzyme and found another lysyl residue, Lys382, was modified. Reductive phosphopyridoxylation of the wild-type and K419R enzymes caused dramatic alteration in allosteric properties. The activator, 3-P-glycerate, and inhibitor, P(i), protected the enzyme from reductive phosphopyridoxylation. The modified enzymes were more active in the absence of 3-P-glycerate and less sensitive to P(i) inhibition.

Partial purification and characterization of invertase isozymes from rice grains (Oryza sativa).

Four invertase isozymes have been isolated from the milky stage rice grains. According to the pH optima, they are classified as one alkaline (IT7) and three acid invertases. The acid invertases are further divided into two soluble forms (IT4 and IT5) and one cell wall-bound (ITb) form which was solubilized in 1 M NaCl. The pH optima of ITb, IT4, IT5 and IT7 are 4.5, 3.5-4.0, 5.0 and 7.0, and the molecular masses are 42, 60, 64 and 260 kDa, respectively. Both IT4 and IT5 were bound to Con A-Sepharose suggesting that these enzymes are glycoprotein. The Km of ITb, IT4, IT5 and IT7 for sucrose are 4.3, 0.9, 12.1 and 70.1 mM, respectively. IT4 and IT5 have a higher Km for raffinose, and the maximum activities are 64% and 27% of that using sucrose as the substrate. IT7 did not hydrolyze raffinose at all. These invertases also exhibit distinct isoelectric points (pI) and different susceptibility to various inhibitors.

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.