JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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H290, H284, H332 and H333 around the CuB site were found while the other, CuB, directly binds to three other histidine resi-
to be required for catalytic function and to act as copper-coordi- dues (H363, H367 and H390) and indirectly binds to H389. We fur-
nating ligands of the mature tyrosinase from A. oryzae23.
ther assume that copper coordination at one site may facilitate
copper binding at the other site. However, the crystal structure of
Moreover, Spritz et al.22 described that human tyrosinase contains
four conserved histidine residues (H363, H367, H389 and H390).
They further showed that the H390A mutant eliminated catalytic
activity, but did not reduce copper binding, whereas the H389
replacement resulted in complete lack of activity. This suggested
that only H389 plays an essential role in copper binding of human
tyrosinase, despite the recognition of H390 as a conserved residue
in all putative copper binding sites22. However, we found that the
H363, H367 and H390 residues of human tyrosinase are likely dir-
ectly involved in copper binding based on evaluations of catalytic
activity and structural modelling. Furthermore, in contrast to these
previous findings, H389 did not abolish the catalytic activity for L-
tyrosine hydroxylation and showed indirect binding with the cop-
per atom at the CuB site. Although we showed that H389 was
indirectly involved in tyrosine hydroxylation reaction, its precise
role at the CuB site requires further investigation.
human tyrosinase is required to identify the crucial residues for
copper binding in catalysing reactions.
Author contributions
Conception and design of the experiments: SH, KHK; Development
of methodology: HN, SJL, HJJ, HWC; Acquisition of the data: HN,
SJL, HJJ, HWC; Analysis and interpretation of data: HN, SJL, SH,
KHK; Writing and review of the manuscript: HN, SH, KHK; Study
supervision: SH, KHK
Disclosure statement
The authors declare no conflict of interest.
Although the CuB site exhibits a higher conservation than CuA,
studies of tyrosinase from different species have shown the
involvement of the conserved histidine residues in copper bind-
ing. The conserved histidine residues around copper ions may
play different roles in stability, flexibility, and additional structure
conformation for the catalytic activity of tyrosinase. According to
structure studies in other species, H54 in tyrosinase from S. casta-
neoglobisporus (TyrSc) was ligated to CuA that is transferred from
the caddie protein through H82, M84 and H97 to the flexible H54
residue, which changes its conformation to coordinate the copper
in the active site34. In B. megaterium tyrosinase (TyrBm), M61 and
M184 residues transfer copper ions to the flexible H60 (in the
same manner as H54 in TyrSc), which positions the copper ion in
the active site35. Furthermore, H251 residues of A. bisporus tyrosin-
ase coordinate CuB in the active site, which consequently causes
CuB flexibility in the active site36. A structural study of tyrosinase
from A. oryzae (TyrAo) showed that the thioether bond between
H94 and C92 is formed only in the presence of copper in the
active site37.
Moreover, it has been proposed that the proton of the
hydroxyl group of monophenols may be removed and transferred
to one of the coordinating histidine residues in the catalytic
mechanism of tyrosinase38,39. The deprotonated substrates are
bound to the active centre of tyrosinase40, and this may be stabi-
lised by the interaction with the conserved histidine residues serv-
ing as a proton acceptor, even though this should be proved by
compelling evidence.
Furthermore, recent studies have identified that an Asn residue
along with a Glu residue is critical for tyrosinase tyrosine hydroxyl-
ation activity at Cu binding sites, showing that polyphenol oxi-
dase, which lacks monophenolase activity, can be transformed to
a tyrosinase by simply introducing an asparagine. A conserved
water molecule activated by asparagine and glutamate is pro-
posed to mediate the deprotonation of the monophenol at the
active site41,42. Another study showed that an alternative set of
four key residues, including a conserved glutamate and phenyl-
alanine, are located around copper active sites with conserved
copper-coordinating histidine residues43. The present study thus
further expands this mechanism by demonstrating the key role of
histidine residues of tyrosinase for its catalytic activity, although
further study may be needed to elucidate the role of each histi-
dine residue in this process.
Funding
The research was supported by National Research Foundation of
Korea Grant of the Korean Government [NRF-2018R1A2B6009246]
and Chung-Ang University Research Scholarship Grants in 2019.
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Thus, we conclude that one of a pair of copper ions, CuA, dir-
ectly binds to three histidine residues (H180, H202 and H211),