Stereospecificity and catalytic function of histidine residues in 4a-hydroxy-tetrahydropterin dehydratase/DCoH.
Three conserved histidines have been shown to be important for the enzymatic activity of 4a-hydroxy-tetrahydropterin dehydratase, a bifunctional enzyme which is involved in regeneration of tetrahydrobiopterin and is also a cofactor (DCoH) for the transcription factor HNF-1alpha. The 4a isomer dependent kinetics of the mutants of rat/human enzyme, H61A, H62A, and H79A, and the effect of diethylpyrocarbonate (DEPC) have been investigated to elucidate the dehydratase mechanism. At pH 6.5 wild-type enzyme is inactivated by DEPC after derivatization of one histidine, shown to be H61 by comparison to H61A. H79 is also derivatized by DEPC at pH 7.0 and above, whereas H62 does not react at any pH. Dehydratase activity of H61A with 4a(R)-hydroxy-6(S)-methyl-tetrahydropterin was not detectable. In contrast, although Km for the enantiomeric 4a(S)-hydroxy-6(R)-methyl-tetrahydropterin was 65-fold higher than with wild-type, kcat was 86% of wild-type. H79A gave complementary results: activity with 4a(S)-hydroxy-6(R)-methyl-tetrahydropterin was undetectable, but 4a(R)-hydroxy-6(S)-methyl-tetrahydropterin had almost normal Km and 75% of wild-type kcat. Replacing H62 with alanine decreased kcat/Km 80- and 60-fold, and kcat to 24% and 89% of wild-type for the 4a(R),6(S)- and 4a(S),6(R)- isomers, respectively. Near neutral pH nonenzymatic dehydration catalyzed by solvated proton had a rate constant of 1.55 x 10(5) M-1 sec-1. A break in the rate versus pH curve at 5.95 was tentatively assigned to protonation of the carbinolamine guanidinium system. The free acid of acetic acid and the imidazolium ion showed general acid catalysis of 18.5 and 1.5 M-1 sec-1, respectively, in dehydrating the neutral carbinolamine. Compared to the later value, dehydratase effective molarity is 11 M. These results are consistent with a dehydratase mechanism in which H61 and H79 act as general acid catalysts for the stereospecific elimination of the 4a(R)- and 4a(S)-hydroxyl groups, respectively. The role of H62 is primarily binding substrate, with an additional component of base catalysis.
Rebrin
I
I
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688, USA.
Thöny
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B
Bailey
S W
SW
Ayling
J E
JE
D000409:Alanine / Q000235:genetics; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002021:Buffers; D002384:Catalysis; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019346:Sodium Acetate; D013237:Stereoisomerism; D013379:Substrate Specificity
D002021:Buffers; D007093:Imidazoles; D011994:Recombinant Proteins; D001708:Biopterins; D019346:Sodium Acetate; D006639:Histidine; C093282:4a-hydroxytetrahydrobiopterin; D006836:Hydro-Lyases; C076392:pterin-4a-carbinolamine dehydratase; D004047:Diethyl Pyrocarbonate; D000409:Alanine