Biochemical characterization of plant biotin-containing enzymes

3-Methylcrotonyl-CoA carboxylase has been purified and characterized from maize leaves. The enzyme is composed of two subunits, a biotin-containing polypeptide of 80 kDa, and a nonbiotin-containing polypeptide of 58 kDa. The native molecular weight of the holoenzyme is estimated at 853,000. The enzyme probably has an [alpha][subscript]6[beta][subscript]6 configuration. The kinetic constants for the substrates of 3-methylcrotonyl-CoA and its pH optimum were determined. The enzyme is allosterically activated by Mg[superscript]2+ and nonessentially activated by monovalent cations. The enzyme is strongly inhibited by acetoacetyl-CoA and by arginyl and sulfhydryl modifying reagents;The effect of K[superscript]+ on 3-methylcrotonyl-CoA carboxylase has been studied. K[superscript]+ affects the binding of ATP and 3-methylcrotonyl-CoA to the enzyme, but does not affect the binding of bicarbonate. The results indicate that K[superscript]+ is involved in the first half-reaction in which the carboxybiotinyl intermediate is formed;Initial velocity studies with competitive inhibitors of soybean 3-methylcrotonyl-CoA carboxylase are consistent with the enzyme following a random Bi Bi Uni Uni Ping Pong mechanism. A rate equation for this mechanism has been deduced;Propionyl-CoA carboxylase activity has been purified from maize leaves. This activity copurified with acetyl-CoA carboxylase activity throughout the purification procedures. Inhibition studies and mixed substrate analyses suggest that propionyl-CoA carboxylase activity detectable in extracts from plants is a side reaction of the acetyl-CoA carboxylase enzyme;Geranoyl-CoA carboxylase has been partially purified and characterized from maize leaves. The kinetic constants for the substrates of the enzyme, geranoyl-CoA, ATP, and bicarbonate, and its pH optimum have been determined. The enzyme occurs ubiquitously through the maize plant. This is the first demonstration of the presence of geranoyl-CoA carboxylase in eukaryotic organisms. A potential metabolic function of this enzyme in the catabolism of monoterpenes is discussed