Aminolevulinate synthase: Lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis

Abstract

Abstract5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-3 13 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5"phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2: 1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5"phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5"phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release

“…40,60 However, the nature of the nucleophile involved in the formation of the adduct in ALAS has yet to be identified. 40 Furthermore, and as observed with murine ALAS2, 29,55,56 since PLP is not a chiral molecule, the positive dichroic bands with maxima at ~330 and 420 nm in the CD spectra of wild-type hALAS2 and XLPP variants indicate an equilibrium between two populations of internal aldimine species with different chiral active site environments.…”

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

“…Previously, we assigned the 420 nm-absorbance maximum to a ketoenamine species, and we utilized fluorescence spectroscopy to assign the 330 nm maximum to a substituted aldamine (8). Moreover, positive dichroic bands with maxima at approximately 330 and 420 nm are detected in the circular dichroism (CD) spectrum of mALAS2 (9,10), and since PLP by itself is not a chiral molecule (10), the two maxima indicate an equilibrium between two populations of internal aldimine species with different chiral active site environments.…”

Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase

“…Previously, it was reported that linking the two ALAS subunits into the single chain dimeric ALAS/ALAS resulted in over 5-fold and 28-fold increases in the k cat and $k_{\text{cat}}/K_{\text{m}}^{\text{SCoA}}$ values, respectively [23]. To determine the individual contribution of the two active sites to the overall steady-state activity of ALAS/ALAS, the steady-state kinetic parameters of the ALAS K313A /ALAS and ALAS/ALAS K313A variants, in which the K313A mutation [27,37] was independently introduced in each of the two active sites, were determined (Table 3). While the K313A mutation in ALAS K313A /ALAS decreased the k cat 2.5-fold, the same mutation in ALAS/ALAS K313A resulted in a 12.6-fold decrease of the k cat value.…”

“…The pTDT12 and pTDT17 expression plasmids (Table 1) were constructed using the pGF27 expression plasmid for the ALAS K313A variant [27] as the starting material for a DNA piece coding for the ALAS K313A mutation. The pGF27 plasmid was digested with Kpn I and Xba I, and the ALAS K313A -encoding fragment was ligated into pTDT4 and pTDT5 digested with the same enzymes.…”