Acetohydroxy acid reductoisomerase of wheat

Abstract

Acetohydroxy acid reductoisomerase (EC 1.1.1.86, AHAR) was purified to a high degree from green shoots of wheat (Triticum aestivum L. cv. Vulcan). The enzyme was localised in the chloroplasts, and activity was at a maximum approximately 4 d after germination. The subunit molecular mass of wheat AHAR was 57 kD and activity of the native enzyme had an elution volume from size exclusion columns that corresponded to a molecular mass of 47 kD. The enzyme did not require the addition of Mg 2+ ions to reaction mixtures for activity. The Km values for (R,S)-2-acetolactate and (R,S)-2-aceto-2-hydroxybutyrate were 91 and 9 mM, respectively, and the corresponding maximum velocities were 430 and 451 mU mg –1 protein. The Km for NADPH was approximately 10 mM when either of the acetohydroxy acids was the other substrate. Preparation of the acetohydroxy acid substrates by hydrolysis of the parent esters in strong base led to the formation of inhibitory by-products. Racemisation of the acetohydroxy acids was detected in assay mixtures.

Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase

“…Consequently, the minimum oligomeric structure of Class I KARIs is dimeric with some enzymes forming dodecamers . However, while plant KARIs are either mono‐ or dimeric, bacterial Class II KARIs (e.g., from E. coli ) form tetramers…”

“…Consequently,t he minimum oligomeric structureo fC lass I KARIs is dimeric [5c] with some enzymesf orming dodecamers. [7] However,w hile plant KARIs are either mono- [8] or dimeric, [5b, 9] bacterialClass II KARIs (e.g.,from E. coli)f orm tetramers. [10] The observed variationsi nt he oligomeric structure may be associated with differences observed in the assembly of ac atalyticallya ctive site upon addition of the cofactors (i.e.,M g 2 + , NADPH)a nd/or substrates.…”

“…[5a, 10a] However,i np lant KARI the Ndomain in the apoenzyme appears to have asignificant level of rotational freedom,b ut upon Mg 2 + andN ADPH binding the Na nd Cd omains are locked into place thus forming the recognition site for substrate binding. [5a] Based on the similarityo ft heir catalytic parameters (i.e., k cat values and Michaelis constants K m for substrates), [4,8,9,11] it appears likely that the structure of the catalytically competent enzyme-substrate complexa nd consequently the reactionm echanism is conserved in all KARIs.In this study,t he reactionm echanismso fE. coli and rice KARI are investigated using ar ange of physico-chemical techniques.…”

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins

“…Discriminant analysis was performed using SPSS statistical software (16.0.0) (IBM Corporation, Somers, NY, USA) on a randomly selected 70% sample and tested using the remainder. Unnormalised data were used: Box's M statistic 15.3 (f(10,3149) = 1.355, p = 0.195). All the amino acids were used in the initial analysis, even where measurements were missing for some samples.…”

“…Although biochemical pathways are nominally the same in substantially all organisms, even closely related species may have enzymes with markedly different properties. For example, an enzyme involved in the synthesis of isoleucine, valine and isoleucine, ketol‐acid reductoisomerase (KARI) purified from two cereals – barley and wheat – showed different characteristics. The kinetic rate constant (K m ) for the wheat enzyme was some ten times greater for acetolactate (a precursor of valine and leucine) than for acetohydroxybutyrate (a precursor of isoleucine), while the ratio of the constants for the barley enzyme was about 3.5 in the opposite direction.…”