32 Landgraf and WolleesThe sequence for the enzyme allowed a better characterization of
the Taq polymerase (5). Given the biochemical, biophysical, and sequence
data, it can be concluded that the two enzyme preparations published
in the original paper (1,5) represented either two different enzymes or
that the smaller protein was a fragment of the constitutive Taq poly-
merase. The characteristic features of the commercially available cloned
Taq polymerase are analogous to those of the latter protein.
The Taq polymerase gene is 2499 nucleotides long and codes for a
protein of 832 amino acids. The theoretical mass is 93,900 Da, which
is in fairly good agreement with the apparent mass of 97,300 Da obtained
from gel electrophoresis. A sequence comparison with polymerase I
from E. coli shows significant similarities in the 5'-3' exonuclease
domain (amino acids 1-410) as well as in the polymerase domain
(amino acids 410-832). Regions to which definite functions could be
assigned in polymerase I are highly conserved in Taq polymerase.
No proofreading activity (3'-5' exonuclease) could be identified in
Taq polymerase (6). There is no homology in Taq polymerase to a
domain ranging from amino acid 300 to 410 in polymerase I, to which
proofreading activity is attributed in the E. coli enzyme.
Taq polymerase catalyzes the DNA-dependent polymerization of
dNTPs. One unit of the enzyme is defined as the amount of enzyme
that will incorporate 10 nmol of radioactively labeled dTTP into acid-
insoluble material at 80°C in 30 min (1). The enzyme could be purified
to a specific activity of 200,000 U/mg protein. As an optimal reaction
temperature, 75-80 ° C was established, at which the incorporation rate is
approx 150 nucleotides/s/enzyme molecule (7). In the M 13 system, incor-
poration rates of <60, 24, 1.25, and 0.25 nucleotides/s/enzyme molecule
were measured at 70, 55, 37, and 22°C respectively (8). Incorporation
rates decrease at temperatures >90°C; the stability of the enzyme also
decreases drastically. The half-life time of the enzyme declines from 130
to 40 to between 5 and 6 min at 92.5, 95, and 97.5°C respectively (7).
Enzyme activity is dependent on bivalent cations. Concentrations
of 2 mM MgCI 2 are optimal. Maximum polymerization rates are obtained
with 0.7-0.8 mM dNTPs. Substrate inhibition is observed at dNTP con-
centrations of 4-6 mM. Monovalent cations also have an effect on the
activity of the enzyme. Optimum conditions are 50 mM KC1, whereas
inhibition is reported at concentrations >75 mM KC1 (8). NaC1, NH4C1,
and NH 4 acetate cannot substitute for KC1 without a decrease in specific