Chemistry Reference
In-Depth Information
Figure 2.5 Structure of the PEI-pyridoxamine systems.
(Reprinted from Ref. 36. Copyright 2002 American
Chemical Society.)
free pyridoxamine rate constant by 6000 times. However, the rate for the polymer in-
creased by only 30-fold. The transaminase enzymes do not use metal ions.
The rate enhancement of the polymer over that of simple pyridoxamine was a steep
function of the length of the alkyl chains added, in polymers with roughly the same
percentage of alkylation and of pyridoxamine attachment. At pH 7.0 and 30
8
C, the
acceleration over the rate with pyridoxamine was 160 for C-1 chains, 180 for C-3,
500 for C-6, 1000 for C-9, 2300 for C-12, and 2500 for the C-15 and C-18 normal alkyl
chains. This chain effect seems unlikely to involve hydrophobic binding of a substrate
as hydrophilic as pyruvic acid. Instead the hydrophobic chains modify the pK
a
softhe
amino groups in the polymer and also create a cavity in which the transamination can
take place in a less than fully aqueous environment.
Our further study on the PEI-pyridoxamine systems revealed that the amination
reaction of pyruvic acid with simple pyridoxamine was buffer catalyzed, but the reac-
tion with our polymer-linked pyridoxamine showed no catalysis by external buffers
[36]. This supported the argument that the acid and base groups of the polymer
were performing catalytic proton transfers in the transamination process. Extrapolating
[buffer] to zero, the PEI-pyridoxamine system was actually 10 000-fold faster than was
simple pyridoxamine with pyruvic acid at pH 7.0. Furthermore, the polymer system
showed saturation effects when titrated with substrates (Michaelis-Menten kinetics).
Laurylation of the polymers both increased k
2
by producing a less aqueous reaction en-
vironment and selectively bound the more hydrophobic substrates, decreasing K
M
.
This resulted in a high rate acceleration for all substrates, and a particularly high
acceleration for hydrophobic substrates. The amination rate of indolepyruvic acid
to tryptophan was enhanced 240 000-fold relative to that with simple pyridoxamine.
To study the effect of polymer size on catalysis [37], pyridoxamine was linked to a
series of PEIs with M
n
= 600, 1800, 10 000, and 60 000, both simply permethylated and
with additional attached dodecyl chains. The polymers were examined in the transa-
mination of pyruvic acid and of phenylpyruvic acid, showing Michaelis-Menten be-
havior. The k
2
and of K
M
determined showed only small variations with polymer size.
Thus, the strong advantage of pyridoxamines attached to the M
n
= 60 000 PEI, relative
to simple pyridoxamine alone, was seen to almost the same extent with the smaller
PEIs.
Notably, the PEI enzyme models do not have a well-defined structure, and each poly-
mer molecule contains more than one coenzyme unit. To make more defined macro-
molecular transaminase models we synthesized several water-soluble poly(amidoa-
