| Abstract
| - 5-Aminolevulinate synthase catalyzes the first step ofthe heme biosynthetic pathway in animals,fungi, and some bacteria. The enzyme belongs to a large family ofenzymes that use pyridoxal 5‘-phosphateas an essential cofactor. We previously analyzed the informationalcontent contained in each residue ofa conserved glycine loop, which we proposed to form part of thecofactor binding site [Gong, J., &Ferreira, G. C. (1995) Biochemistry 34, 1678−1685].We found that Gly-142 and −144 contain highinformational content, and we identified G144A, G144S, G144T, and G142Cas functional mutants. Here,the catalytic parameters, cofactor affinities, and spectral andthermostability properties of these four glycinemutants are determined to examine the function of the glycine loop.In addition, computer models of theglycine loops from the wild-type and mutant enzymes were generated,using glycogen phosphorylase bas the structural template. G144A, G144S, G144T, and G142Cdisplayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and15-fold increases, respectively, in thedissociation constant value for binding of the cofactor. While theturnover numbers for G144A, G144S,G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value,respectively, the Km values forboth substrates remained unchanged, with the exception of the G142CKmGly, which showed a4-foldincrease. The UV−visible and CD spectra of Gly-144 mutants weresimilar to those of the wild type;however, the spectral properties of G142C suggest that this mutantbinds the cofactor in a different modeat the active site. G144A, G144S, G144T, and G142C were also foundto be less stable than the wild-type enzyme, with the thermotransition temperature,T1/2, determined to be 3.5, 3, 3.5, and 5°C, respectively,lower than that of the wild-type enzyme. Collectively, computermodeling of the wild-type and mutantforms of the ALAS glycine loop and biochemical and spectroscopiccharacterization of G144A, G144S,G144T, and G142C strongly suggest that the conserved glycine loop in5-aminolevulinate synthase is apyridoxal 5‘-phosphate cofactor binding motif.
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