Biochemical characterization of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients

The biochemical properties of mutant phenylalanine hydroxylase (PAH) enzymes and clinical characteristics of hyperphenylalaninaemic patients who bear these mutant enzymes were investigated. Biochemical characterization of mutant PAH enzymes p.D143G, p.R155H, p.L348V, p.R408W and p.P416Q included determination of specific activity, substrate activation, V max, K m for (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄), K d for BH₄, and protein stabilization by BH₄. Clinical data from 22 patients either homozygous, functionally hemizygous, or compound heterozygous for the mutant enzymes of interest were correlated with biochemical parameters of the mutant enzymes. The p.L348V and p.P416Q enzymes retain significant catalytic activity yet were observed in classic and moderate PKU patients. Biochemical studies demonstrated that BH₄ rectified the stability defects in p.L348V and p.P416Q; additionally, patients with these variants responded to BH₄ therapy. The p.R155H mutant displayed low PAH activity and decreased apparent affinity for l-Phe yet was observed in mild hyperphenylalaninaemia. The p.R155H mutant does not display kinetic instability, as it is stabilized by BH₄ similarly to wild-type PAH; thus the residual activity is available under physiological conditions. The p.R408W enzyme is dysfunctional in nearly all biochemical parameters, as evidenced by disease severity in homozygous and hemizygous patients. Biochemical assessment of mutant PAH proteins, especially parameters involving interaction with BH₄ that impact protein folding, appear useful in clinical correlation. As additional patients and mutant proteins are assessed, the utility of this approach will become apparent.