ACTIVE-SITE OF 5-AMINOLEVULINATE SYNTHASE RESIDES AT THE SUBUNIT INTERFACE - EVIDENCE FROM IN-VIVO HETERODIMER FORMATION

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the he me biosynthetic pathway of animals, fungi and some bacteria. It functi ons as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 h as been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sc i. 2, 1959-1965], while arginine 149, a conserved residue among all kn own 5-aminolevulinate synthase sequences, is essential for function [G ong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whethe r each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interf ace (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically i nactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A(-) Escherichia coli strain, whic h can only grow in the presence of 5-aminolevulinate or when it is tra nsformed with an active 5-aminolevulinate synthase expression plasmid, the hem A(-) E. coli strain acquired heme prototrophy. The purified K 313A/R149A heterodimer mixture exhibited K-m values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA seque ncing of four Saccharomyces cerevisiae 5-aminolevulinate synthase muta nts, which lack ALAS activity but exhibit enzymatic complementation, r evealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthas e active site is located at the subunit interface and contains catalyt ically essential residues from the two subunits.