Kinetic and spectroscopic evidence of negative cooperativity in the action of lysine 2,3-aminomutase

Abstract

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-β-lysine, a component of a number of antibiotics. The reaction requires the cofactors S-adenosyl-L-methionine (SAM), pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster. LAM is a founding member of the radical SAM superfamily of enzymes. LAM is highly specific for L-lysine and will not accept most other amino acids as substrates. L-alanine and L-2-aminobutyrate at 0.2 M react as substrates for LAM at, respectively, 5 × 10(-6) and 8 × 10(-5) times the rate with saturating L-lysine. Saturating ethylamine accelerates the L-alanine reaction 70-fold, and saturating methylamine accelerates the L-2-aminobutyrate reaction 47-fold. The primary amines binding at the active site of LAM with L-alanine or L-2-aminobutyrate simulate L-lysine. The steady-state kinetics of the reaction of L-alanine + ethylamine displays negative cooperativity with respect to L-alanine. The second-order rate constant for production of β-alanine in the reaction of L-alanine and saturating ethylamine is 0.040 M(-1) s(-1), which is 2 × 10(-5) times the value of k(cat)/K(m) for the reaction of L-lysine. When L-lysine is at a concentration 1/16th of K(m), the lysyl-free radical intermediate is hardly detectable by EPR; however, the addition of L-alanine at high concentration (0.2 M) enhances free radical formation, and the addition of ethylamine further enhances radical formation. These facts complement the kinetic observations and support negative cooperativity in the reaction of L-alanine as a substrate for LAM. Present results and independent evidence support negative cooperativity in the reaction of L-lysine as well.

Figure 1

Initial rates of 2,3-aminomutase activity of LAM, with L-alanine + ethylamine simulating the substrate L-lysine. Initial rates of β-alanine formation are plotted versus L-alanine concentrations. Rates were measured by the procedure in Experimental Procedure at pH 8.0 and 37 °C in the presence of 0.2 M ethylamine. Initial rates were measured in quadruplicate. Equation 5 was recast into the form v/[Ala] = k[E_o][Ala]/(K_S + [Ala]) and fitted to the data using the program Hypero of Cleland. Values of k and K_S are given in Table 2.

Figure 2

EPR spectra of product radical formation at the active site in LAM. Procedure is described in the Experimental section. The black spectrum is that of the product-related radical 3 in Scheme 1 elicited by the natural substrate L-lysine at 120 mM in the steady state. The dashed spectrum is that obtained upon substitution of L-alanine for L-lysine. The weak orange signal was obtained with L-lysine at a concentration 1/16 the K_m for L-lysine, the green spectrum upon addition of 0.2 M L-alanine, and the red spectrum upon further addition of 0.2 M ethylamine. The blue spectrum was obtained upon substitution of n-propylamine for ethylamine in the preceding experiment.

Scheme 1

Scheme 2