Relationship between Kinetic Properties of Mutant Enzyme and Biochemical and Clinical Responsiveness to Biotin in Holocarboxylase Synthetase Deficiency

Abstract Holocarboxylase synthetase (HCS) deficiency is a metabolic disorder that causes a biotin-responsive multiple carboxylase deficiency. We analyzed the kinetic properties of seven mutant HCS proteins. Two of these enzymes harbored mutations within the putative biotin-binding region of HCS and showed elevated Km values for biotin compared with that of the wild-type form (Km mutant; Gly581Ser: 45 times, delThr610: 3 times). The remaining five mutations (Arg183Pro, Leu216Arg, Leu237Pro, Val333Glu, and Val363Asp) were located outside the biotin-binding region. The enzymes containing these mutations showed normal or low Km values for biotin (non-Km mutant). Symptoms of patients who have the non-Km mutants, as well as those of patients who have the Km mutants, responded to biotin therapy. This is probably because the Km value for biotin of normal HCS is higher than the physiologic concentration of biotin in human cells. The Vmax values of all mutant HCS proteins were considerably decreased, but to a variable degree. The responsiveness to biotin supplementation of propionyl-CoA carboxylase activity in cultured cells bearing the mutations correlated well with the degree of reduction in the Vmax of HCS. Patients who have mutant HCS proteins with lower Vmax showed poorer clinical and biochemical responses to biotin therapy. These observations suggest that the reduction of Vmax is an essential factor for pathophysiology and prognosis of HCS deficiency under treatment with large amounts of biotin. The determination of HCS genotype can be valuable for characterizing the clinical phenotype in HCS deficient patients. Main Holocarboxylase synthetase (HCS: EC 6.3.1.10) is an enzyme that catalyzes biotin incorporation into multiple carboxylases. In humans, there are three mitochondrial carboxylases, namely pyruvate carboxylase, propionyl-CoA carboxylase (PCC), and methylcrotonyl-CoA carboxylase. Acetyl-CoA carboxylase (ACC) is the only known cytosolic carboxylase. Biotin is the prosthetic group of these carboxylases. A deficiency in HCS has been shown to be the cause of the early-onset type of biotin-responsive multiple carboxylase deficiency (McKusick 253270). Most patients with HCS deficiency show acute episodes of metabolic acidosis and characteristic organic aciduria due to the decreased activity of multiple carboxylases before a few months of age (1, 2). Symptoms of the patients include tachypnea, feeding difficulties, and seizures, which may lead to coma or even death. Some patients become symptomatic in the later infantile period, at the age of several months to years (3–6). All HCS-deficient patients so far reported have responded to biotin administration, although in some patients the response was only partial as manifested by continued excretion of abnormal metabolites in the urine (7–9). Developmental abnormalities have also been reported in some cases in spite of high-dose biotin therapy (7, 9). Burri et al. characterized HCS activity in cultured cells from seven patients with considerable differences in disease severity (10). They showed that the Vmax values of patients' HCS were lower than the normal mean and were similar in all except one patient, and that the Km values for biotin were elevated to various degrees. From their clinical and kinetic observations, they concluded that onset of the disease and its responsiveness to biotin administration is governed by the degree of abnormality in the Km of HCS. Suormala et al. investigated the effects of biotin concentration on carboxylase activities in fibroblasts in five HCS-deficient patients (5). The carboxylase activities of all patients normalized when the biotin concentration was increased to 10 000 nM. In carboxylase reactivation studies, although the kinetics of PCC activation varied greatly, all cells restored their PCC activity to the normal or nearly normal level (>87%). Reactivation of PCC activity in relation to time and biotin concentration correlated well with the severity and age at onset of the illness in four patients. These results were consistent with the original “Km mutant theory” proposed by Burri et al. (10). Human HCS cDNA has been cloned (11, 12) and has enabled investigation of HCS at the molecular level. The protein encoded by this cDNA is 726 amino acids in length and has a homologous region (aa 448–701) to Bir A, the biotin apo-carboxyl carrier protein ligase of Escherichia coli (11). This portion of human HCS is thought to be the putative biotin-binding region. In a previous report, we characterized two mutations, Val550Met and Leu237Pro (13). The former was found in a patient with mild clinical phenotype and the latter in patients with severe form. An expression study demonstrated that the Km of the Val550Met HCS mutant was higher than, but the Km of the Leu237Pro-mutant was the same as, that of the wild-type enzyme (13). Thus, we questioned whether the “Km mutant theory” could be applied to all patients with HCS deficiency, and proposed that not only the Km of HCS for biotin, but also the Vmax, is an important factor in determining the severity of symptoms and their responsiveness to biotin therapy (13). We tested our hypothesis in the present study by analyzing the kinetics of HCS mutants, including a newly identified mutation. We also examined the relationship between the kinetic characteristics of HCS mutants and the clinical and biochemical features of the HCS deficient patients. METHODS Patients. Nine HCS-deficient patients were examined in the present study. Patient AD was born to healthy consanguineous Turkish parents (5, 14). On the 2nd day of life, she showed metabolic acidosis. The response of symptoms to biotin treatment was good, although small amounts of 3-hydroxyisovalerate were detected in the urine with 20 to 40 mg/day of biotin. Patient HR is a girl of Lebanese descent born to consanguineous parents (15). After the first severe symptoms appeared at the age of 20 mo, she showed recurrent attacks of vomiting associated with tachypnea and lethargy. Diagnosis was achieved at the age of 5 y and biotin administration was started (10 mg/day). The clinical manifestations of patients UW and KT have been described earlier as patients 1 and 4b, respectively (16). Briefly, patient UW developed metabolic acidosis on the 1st day of life and oral biotin administration (10 mg/day) led to remarkable improvement. With 40 mg/day of biotin, she has remained asymptomatic but urinary organic acid analysis revealed slightly elevated excretion of 3-hydroxyisovalerate. The elder sister of KT presented multiple carboxylase deficiency on the 2nd day of life (2). Consequently, the mother was given biotin in the last trimester of pregnancy since a prenatal diagnosis predicted KT to be affected (8). Biotin treatment was implemented immediately after the birth. However, lactate and 3-hydroxyisovalerate in the urine of KT were elevated even with high-dose biotin therapy (40–100 mg/day), a (...truncated)