• Bulletin of the Korean Chemical Society
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• v.34 no.2
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• pp.669-672
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• 2013

• Applied Biological Chemistry
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• v.40 no.4
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• pp.277-282
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• 1997

Acetolactate synthase (ALS) was partially purified from Escherichia coli MF2000/pTATX containing Arabidopsis thaliana ALS gene. The partially purified ALS was examined for its sensitivity toward various modifying reagents such as iodoacetic acid, iodoacetamide, N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (PCMB), and phenylglyoxal. It was found that PCMB inhibited the enzyme activity most strongly followed by DTNB and NEM. Since iodoacetic acid did not compete with substrate pyruvate, it appeared that cysteine is not involved in the substrate binding site. On the other hand, the substrate protected the enzyme partly from inactivation by phenylglyoxal, which might indicate interaction of arginine residue with the substrate. The partially purified enzyme was inhibited by end products, valine and isoleucine, but not by leucine. However, the ALS modified with PCMB led to potentiate the feedback inhibition of all end products. Additionally, derivatives of pyrimidyl sulfur benzoate, a candidate for a new herbicide for ALS, were examined for their inhibitory effects.

• Korean Journal of Weed Science
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• v.16 no.2
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• pp.132-139
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• 1996

Acetolactate synthase(ALS) activity was determined in germinating seedlings of two rice cultivars treated with cinosulfuron [3-(4,6-dimethoxy-1,3,5-triazin-2-yl)-1-[2-methoxyethoxy)-phenylsulfonyl]-urea]. IR 74(Indica type) was more tolerant than Hwajinbyeo(Japonica type) under various rates of cinosulfuron applied at the pregermination stage. In vitro response of ALS activity in the two rice cultivars was similar to $I_{50}$ values(cinosulfuron concentration required for 50% inhibition of ALS activity) of about 23ppb. In vivo, ALS activity of IR 74 increased as the seedlings grew, but that of Hwajinbyeo dropped at 5 days after 10ppm cinosulfuron treatment and shoot growth of Hwajinbyeo lagged at 4 to 5 days after herbicide treatment. ALS activity and shoot growth of Hwajinbyeo was resumed from cinosulfuron-induced inhibition at 6 days after cinosulfuron treatment. The differential response of ALS activity in two different rice cultivars against cinosulfuron may not be due to difference of ALS sensitivity, but rather due to different metabolic inactivation rates of cinosulfuron.

• Journal of the Korean Chemical Society
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• v.41 no.6
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• pp.304-312
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• 1997

Acetolactate synthase(ALS) is the common enzyme in the biosynthetic of valine, leucine, and isoleucine, and is the target of several classes of structually unrelated herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. In an effort to develop new and desirable herbicides, we have synthesized 4,6-dimethoxypyrimidine derivatives, and examined their inhibitory activities on pea ALS. The most active compound was found to be K11570 and $IC_{50}$ value for K11570 was 0.2 ${\mu}M.$ The inhibition of pea ALS by K11570 was biphasic, showing increased inhibition with incubation time. The K11570 showed mixed-type inhibition with respect to substrate pyruvate. Dual inhibition analysis of K11570 versus sufonylurea herbicide Ally and feedback inhibitor leucine revealed that three inhibitors were competitive for binding to ALS. The arginine modified enzyme showed decreased inhibition by K11570, sufonylurea Ally, and leucine, in constrast to, tryptophan modification did not affect on the sensitivity of ALS to the inhibitors.

• BMB Reports
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• v.32 no.4
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• pp.393-398
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• 1999

Acetolactate synthase (ALS) catalyzes the first reaction common to the biosynthesis of L-valine, L-leucine, and L-isoleucine. ALS is the target site of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Two forms of ALS (ALS I and ALS II) which have different affinity for Heparin have been separated from etiolated pea seedlings. The substrate saturation curves of both ALS I and ALS II were hyperbolic in contrast to previous reports. The two forms of ALS showed significant differences in their physical and kinetic properties. The values of $K_m$ for ALS I and ALS II were 9.0 mM and 4.8 mM, respectively. The pI values for ALS I and ALS II were determined to be 5.3 and 5.75 by isoelectric focusing, respectively. The native molecular weights for ALS I and ALS II obtained by nondenaturing gel electrophoresis and activity staining were 124 and 244 kDa, respectively. They also exhibited different sensitivity to feedback inhibition by end-product amino acids and inhibition by Cadre, an imidazolinone herbicide.

• BMB Reports
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• v.31 no.3
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• BMB Reports
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• v.36 no.5
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• pp.456-461
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• 2003

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target site for several classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. Two forms of ALS (designated ALS I and ALS II) were separated from barley shoots by heparin affinity column chromatography. The molecular masses of native ALS I and ALS II were determined to be 248 kDa and 238 kDa by nondenaturing gel electrophoresis and activity staining. Similar molecular masses of two forms of ALS were confirmed by a Western blot analysis. SDS-PAGE and Western blot analysis showed that the molecular masses of the ALS I and ALS II subunits were identical - 65 kDa. The two ALS forms exhibited different properties with respect to the values of $K_m$, pI and optimum pH, and sensitivity to inhibition by herbicides sulfonylurea and imidazolinone as well as to the feedback regulation by the end-product amino acids Val, Leu, and Ile. These results, therefore, suggest that the two ALS forms are not different polymeric forms of the same enzyme, but isozymes.

• BMB Reports
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• v.28 no.1
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• pp.40-45
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• 1995

Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both ${\alpha}$-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was $2480\;M^{-1}{\cdot}min^{-1}$. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both ${\alpha}$-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was $15,000\;M^{-1}{\cdot}min^{-1}$, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by ${\alpha}$-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was $1480\;M^{-1}{\cdot}min^{-1}$ and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.

• BMB Reports
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• v.30 no.4
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• pp.274-279
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• 1997

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branchedchain amino acids, valine, leucine, and isoleucine. ALS is the target site for several structually diverse classes of herbicides including sulfonylureas, imidazolinones. and triazolopyrimidines. We have purified ALS from etiolated barley shoots to homogeneity. The five major purification steps are ammonium sulfate fractionation, DEAE anion exchange, hydroxylapatite, Bio-Gel A gel filtration, and low pressure Mono-Q chrornatoqraphy. Approximately 170-fold purification was achieved and the yield was 0.45% of initial activity in the crude extract. Both SDS-PAGE and Western blot analysis showed a single polypeptide of ALS with an apparent molecular mass of 64 kDa. The result of nondenaturing gel electrophoresis with activity staining indicated that the molecular mass of its native form is approximately 225 to 250 kDa. The values of $K_m$ for pyruvate. pl. and optimum pH of ALS were determined to be 2.0 mM, 5.2. and 7.0. respectively Feedback inhibition studies showed that ALS is more susceptible to leucine than valine. And $IC_{50}$ value of Cadre, a class of irnidazolinones, is about $1.5\mu{M}$ for ALS.

• BMB Reports
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• v.28 no.6
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• pp.471-476
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• 1995

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target enzyme for several classes of structually diverse herbicides. We have synthesized 4,6-dimethoxypyrimidine derivatives as ALS inhibitors, and their inhibitory activities on barley ALS were determined. $IC_{50}$ values for the derivatives are 0.2~200 ${\mu}m$. K11570, the most potent ALS inhibitor with $IC_{50}$ of 0.2 ${\mu}m$, showed mixed-type inhibition with respect to substrate pyruvate, and the progress curves for ALS inhibition by K11570 indicated that the amount of inhibition increased with time. Inhibition-competition experiments were carried out and indicated that three different classes of inhibitors, K11570, a sulfonylurea Ally, and leucine, bind to ALS in a mutually exclusive manner. Chemical modification of tryptophanyl and tyrosyl residues of ALS decreased the sensitivity of ALS to K11570, while cysteine modification did not affect the sensitivity. These results suggest that tryptophanyl and tyrosynyl residues are probably located at or near the inhibitor binding site.