Enzymic O-sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A-44.
The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Saitama.
R
R
Muramatsu
E
E
Nukui
A
A
Sukesada
S
S
Misawa
Y
Y
Komatsu
T
T
Okayama
K
K
Wada
T
T
Morikawa
H
H
Hayashi
K
K
Kobashi
D000595:Amino Acid Sequence; D000991:Antithrombins / Q000378:metabolism; D001483:Base Sequence; D005051:Eubacterium / Q000201:enzymology*; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000991:Antithrombins; D006629:Hirudins; D009838:Oligodeoxyribonucleotides; D013431:Sulfates; D014443:Tyrosine; D015238:Sulfotransferases