THE REPLACEMENT OF TRP392 BY ALANINE INFLUENCES THE DECARBOXYLASE CARBOLIGASE ACTIVITY AND STABILITY OF PYRUVATE DECARBOXYLASE FROM ZYMOMONAS-MOBILIS/

The bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (PDC) from Zymomonas mobil is was changed to alanine which is found in the equivalent position of PDC from yeast, The mutation reduced the decarboxylase activity towar ds pyruvate by a factor of two (60-70 U/mg), whereas the K-m (1.1 mM i n Mes/KOH buffer) remains unchanged compared with the wild-type enzyme . The apparent K-m, for thiamine diphosphate (thiamin-P-2) in the pres ence of 5 mM MgSO4 was increased by a factor of 10 (84 mu M in Mes/KOH buffer) and the tetrameric mutant protein was less stable, as indicat ed by urea denaturation experiments. The mutation enhanced the carboli gase activity of the enzyme towards benzaldehyde by a factor of four. The resulting alpha-hydroxyketone was identified as (R)-phenylacetylca rbinol.