Peroxidase catalysed aerobic degradation of 5,6,7,8‐tetrahydrobiopterin at physiological pH

Abstract

Theaerobicdegradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed byperoxidase(EC 1.1 1.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3 H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(hH)pterin, and this finally rearranges t o 7,g-dihydroxanthopterin.Spectroscopic evidence (ultraviolet, ' H NMR and I3C N MR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3 H)pterin.The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a K,,, of 16.3 pM and k,,, of 22.5 s-I.The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6 R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural ( 6 s ) isomer.By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (hR)-'I,X-dihydro(6H)biopterin K, = 1.3 pM and k,,, = 22.8 s -' ; with (6S)-7,8-dihydro(6H)biopterin K,,, = 13.5pM and k,,, = 51.6s-'; and with (hRS)-7,%dihydro(6H)neopterin K, = 19.2pM and k,,, = 1 2 6 s y ' . (1) is the natural cofactor for the enzymes that hydroxylatc phenylalanine, tyrosine and tryptophan [l] in the presence of oxygen. The cofactor is oxidised to qui~onoid-7,8-BH~ ( 2 ) which is reduced enzymically by dihydropteridine reductase (EC 1.6.99.7) in the presence of NADH back to BH4 and restores the cofactor for further hydroxylation reactions. Whereas simple pterins, e.g. quinonoid-7,8-6-MPHz, which are effective substrates for dihydropteridine reductase, behave normally during kinetic rneasuremcnts, the oxidised natural cofactor ( 2 ) does not. Simple pterins give linear initial rate plots in the standard assay procedure for the uncoupled rcaction (i.e. without hydroxylase) when the quinonoid-7,8-dihydro(6H)-pterin is gencrated from the 5,6,7,8-tetrahydropterin by peroxidase together with hydrogen peroxide [2] or oxygen [3]. When using the same procedure, quinonoid-7,8-BH~ (2) gives biphasic initial rate plots consisting of a fast linear trace followed by a slower one from which two sets of kinetic parameters ( K , and V) can be derived. This is true also [or 5,6,7,8-Tetrahydrobiopterin (BH,)

Myocardial ischemia results in tetrahydrobiopterin (BH 4 ) oxidation with impaired endothelial function ameliorated by BH 4

“…3). The BH 2 can be converted back to BH 4 by dihydrofolate reductase (DHFR) in an NADPH dependent manner and the PH 2 can subsequently breakdown to XPH 2 (40). Although the activity of DHFR has not been measured in the ischemic heart, we observed that postischemic BH 2 administration was totally ineffective in increasing CF and did not replete BH 4 .…”

The auto‐oxidation of tetrahydrobiopterin

“…The ultraviolet spectrum of BH2 in 0.1 M phosphate buffer, pH 6.8 has three broad peaks centered at 230 nm (E, 'It has been reported that these patients excrete dihydroxanthopterin which could be derived from PHz [21,[25][26][27][28][29]. (Fig.1A).…”

“…242,3934-39431 identified the stable product produced in 0.1 M phosphate pH 6.8, as 7,s-dihydrobiopterin. However, Armarego et al [Armarego, W. L. F., Randles, D. and Taguchi, H. (1983) Eur. J. Biochem.…”

“…Furthermore, Armarego et al [21] demonstrated that PH2 was the major product of the autooxidation of BH4 at room temperature (25°C) in 0.09 M Tris pH 7.6'. In view of this result, Armarego et al [15, 211 questioned the identification of BH2 as the major product of the earlier oxidation performed at pH 6.8 (described in the preceding paragraph) especially since the ultraviolet spectra of PH2 and BH2 are nearly indistinguishable at neutral pH [21]. They furthermore stated that at physiological pH the unstable qBH2 rearranges with the loss of the alkyl side-chain to form PHI and does not tautomerize to form BH2 [15, 211. However, this conclusion of Armarego et al [15] is inconsistent with two observations from our laboratory.…”

“…boiling at pH 7.5 [19] or autoclaving at 121°C at pH 4.0 [20]. Furthermore, Armarego et al [21] demonstrated that PH2 was the major product of the autooxidation of BH4 at room temperature (25°C) in 0.09 M Tris pH 7.6'. In view of this result, Armarego et al [15, 211 questioned the identification of BH2 as the major product of the earlier oxidation performed at pH 6.8 (described in the preceding paragraph) especially since the ultraviolet spectra of PH2 and BH2 are nearly indistinguishable at neutral pH [21].…”