Kinetic studies of lysine-sensitive aspartate kinase purified from maize suspension cultures

Steady state substrate kinetics and feedback regulation properties were determined for lysine-sensitive aspartate kinase (AK) purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures. Two AK isoforms (AK Early and AK Late) were separated by two passages through an anion exchange column as the final steps in a procedure giving 1200-fold purification. Kinetic properties were determined for the major AK Late eluting isoform. Assays were conducted at the pH activity maximum (8.0) and with excess Mg²⁺ to favor a two-substrate reaction involving aspartate and complexed MgATP. AK catalyzed a sequential reaction in which MgATP and aspartate both bind to the enzyme complex before the ADP and aspartyl-phosphate products are released. The K_m value calculated for MgATP was 0.43 millimolar and for aspartate was 1.04 millimolar. Cooperativity in substrate binding was not observed and was not induced by lysine. The lysine concentration required for 50% inhibition of AK activity was 7 micromolar. An apparent Hill coefficient of 1.4 indicated a minimum of two lysine-binding sites on the active AK complex. At nonsaturating substrate concentrations, lysine inhibition was characteristic of an S-parabolic, I-parabolic noncompetitive allosteric inhibitor. The parabolic inhibitor replot, Hill coefficients > 1, and the lack of substrate cooperativity were consistent with a model for multiple lysine-binding sites per active AK subunit. Similar kinetic properties were observed for the AK Early isoform.     

Selection and characterization of sethoxydim- tolerant maize tissue cultures

`Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO₃⁻ incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [¹⁴C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [¹⁴C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

Characterization of Maize Acetyl-Coenzyme A Carboxylase

Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.     

Expression of the Acc1 Gene-Encoded Acetyl-Coenzyme A Carboxylase in Developing Maize (Zea mays L.) Kernels

A mutation (Acc1-S2) in the structural gene for maize (Zea mays L.) acetyl-coenzyme A carboxylase (ACCase) that significantly reduces sethoxydim inhibition of leaf ACCase activity was used to investigate the gene-enzyme relationship regulating ACCase activity during oil deposition in developing kernels. Mutant embryo and endosperm ACCase activities were more than 600-fold less sensitive to sethoxydim inhibition than ACCase in wild-type kernel tissues. Moreover, in vitro cultured mutant kernels developed normally in the presence of sethoxydim concentrations that inhibited wild-type kernel development. The results indicate that the Acc1-encoded ACCase accounts for the majority of ACCase activity in developing maize kernels, suggesting that Acc1-encoded ACCase functions not only during membrane biogenesis in leaves but is also the predominant form of ACCase involved in storage lipid biosynthesis in maize embryos.