Ability of tetrahydrobiopterin analogues to support catalysis by inducible nitric oxide synthase: formation of a pterin radical is required for enzyme activity.

Abstract:

:Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H(4)B) or tetrahydrobiopterin analogues (5-methyl-H(4)B and 4-amino-H(4)B), and the ability of bound 5-methyl-H(4)B and 4-amino-H(4)B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H(4)B forms a very stable radical, 5-methyl-H(3)B(*), that accumulates in the arginine reaction to approximately 60% of the HDiNOS concentration and decays approximately 400-fold more slowly than H(3)B(*) (0.0003 vs 0.12 s(-1)). The amount of radical (5-methyl-H(3)B(*) or H(3)B(*)) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H(4)B-saturated FLiNOS and HDiNOS is similar to that when H(4)B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and (*)NO. A pterin radical was not observed with 4-amino-H(4)B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H(4)B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H(4)B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO(-) rather than (*)NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H(4)B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Hurshman AR,Krebs C,Edmondson DE,Marletta MA

doi

10.1021/bi035491p

subject

Has Abstract

pub_date

2003-11-18 00:00:00

pages

13287-303

issue

45

eissn

0006-2960

issn

1520-4995

journal_volume

42

pub_type

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