Vitamin B6 Enzyme Models - Artificial Enzymes - page 49

Figure 2.3 Proposed model for the semisynthetic RNAse-S complex
incorporating a pyridoxal-based amino acid residue. The S-peptide
fragment is highlighted, illustrating the location of pyridoxal (Pal 8 ) and
the general acid-base pair. (Reprinted with permission from Ref. 28.
Copyright 1994 American Chemical Society.)

In an alternative approach to generating the polypeptide-vitamin B6 system Diste-
fano and co-workers [31] employed a family of small lipid-binding proteins to which
pyridoxamine could be covalently attached through a disulfide linkage. Initially they
utilized the adipocyte lipid-binding protein (ALBP, Figure 2.4), which has a cysteine
residue (Cys 117 ) [32]. After attachment of pyridoxamine, the resulting ALBP-pyridox-
amine conjugate was able to reductively aminate various

a

-keto acids to amino acids,
with enantioselectivities ranging from 0 to 94% ee. The reaction rates of these reac-
tions were not, however, significantly faster than those involving free pyridoxamine.
This suggested that the protein cavity was functioning as a chiral environment that
controls the facial selectivity of the protonation of the aldimine intermediate without
forming specific interactions with the bound pyridoxamine cofactor to accelerate the
reaction.
In subsequent studies, Distefano et al. utilized a different fatty acid-binding protein,
i.e. intestinal fatty acid-binding protein (IFABP) [33]. The wild-type IFABP has no Cys
inside the cavity, and thus it is an ideal template to introduce single Cys residues at
different positions. Cys mutations at various positions were introduced using site-di-
rected mutagenesis. By varying the position of the cofactor attachment, and thereby
changing the microenvironment of the “active site“, the rate, enantioselectivity, and
substrate selectivity of the protein-pyridoxamine conjugate were modulated.
Further studies on these protein-pyridoxamine conjugates were devoted to the turn-
over behaviors, metal ion effects, and effects of pyridine quaternization [34]. As many