Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase

Abstract

A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH‐cytochrome b5 reductase gene

“…To date, 14 mutations in the b5R gene have been detected in unrelated patients with hereditary methemoglobinemia; five missense mutations (Arg 57 Gln, Val 105 Met, Leu 148 Pro, Glu 212 Lys, and Thr 116 Ser [polymorphism] ) have been found in patients with type I, three missense mutations (Ser 127 Pro, Cys 203 Arg, and Pro 95 His), two nonsense mutations (Arg 218 Stop and Tyr 42 Stop), two deletion mutations (Phe 298, del 3 bp and Met 272, del 3 bp), and two splicing mutations (the 5′ splice site of exon 5 and the 3′ splice site of exon 9) have been found in patients with type II ( Fujimoto et al , 1993 ; Jenkins & Prchal, 1996, 1997; Katsube et al , 1991 ; Kobayashi et al , 1990 ; Manabe et al , 1996 ; Mota et al , 1995 ; Shirabe et al , 1994 , 1995, 1992; Yubisui et al , 1991 ). Type II mutant enzymes with Ser 127 Pro ( Yubisui et al , 1991 ) and Phe 298 del ( Shirabe et al , 1994 ) and type I enzymes with Arg 57 Gln ( Shirabe et al , 1992 ), Val 105 Met ( Shirabe et al , 1992 ) and Leu 148 Pro ( Nagai et al , 1993 ) have been synthesized in E. coli and characterized. All these mutant enzymes have relatively dynamic amino acid changes such as from basic to acidic or from hydrophobic to hydrophilic amino acid residues, and showed reduced k cat / K m values compared with that of the wild type, indicating impaired catalytic activities of enzymes.…”

“…Assay of enzyme activity was performed with potassium ferricyanide or recombinant human erythrocyte cytochrome b 5 expressed in E. coli ( Nagai et al , 1993 ) as electron acceptors. NADH‐ferricyanide activity was measured at 25°C in 10 m m potassium phosphate (pH 7.5) and NADH‐cytochrome b 5 activity was assayed at 25°C in 5 m m Tris‐HCl (pH 7.5), as described previously ( Yubisui & Takeshita, 1980).…”

Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses

“…Based on enzyme activity alone, one might expect carrier animals with about half normal enzyme activity to have methemoglobinemia, but they do not. This discrepancy can be explained if the abnormal enzyme present in deficient canine erythrocytes is unstable, as has been demonstrated in Cb 5 R‐deficient humans 83. The normal life span of canine erythrocytes is about 100 days in the circulation1 and, presumably, the erythrocyte life span is normal in Cb 5 R‐deficient dogs as it is in deficient humans.…”

Congenital Methemoglobinemia: A Rare Cause of Cyanosis in the Newborn—A Case Report

“…7,14 Type 2 congenital methemoglobinemia is more pervasive and is associated with a generalized systemic deficiency affecting a multitude of tissues, particularly the central nervous system. 14 -16 After additional study with a more sensitive assay, type 3 hereditary enzymopenic methemoglobinemia was shown by Nagai et al 17 to be virtually identical to type 1. As such, an independent type 3 classification was proved to be superfluous and is not currently used.…”