Structure-Function Analysis of Human Glucose-6-phosphatase, the Enzyme Deficient in Glycogen Storage Disease Type 1a

Abstract

Glucose-6-phosphatase (G6Pase) is the enzyme deficient in glycogen storage disease type 1a, an autosomal recessive disorder. We have previously identified six mutations in the G6Pase gene of glycogen storage disease type 1a patients and demonstrated that these mutations abolished or greatly reduced enzymatic activity of G6Pase, a hydrophobic protein of 357 amino acids. Of these, four mutations (R83C, R295C, G222R, and Q347X) are missense and one (Q347X) generates a truncated G6Pase of 346 residues. To further understand the roles and structural requirements of amino acids 83, 222, 295, and those at the carboxyl terminus in G6Pase catalysis, we characterized mutant G6Pases generated by near-saturation mutagenesis of the aforementioned amino acids. Substitution of Arg-83 with amino acids of diverse structures including Lys, a conservative change, yielded mutant G6Pase with no enzymatic activity. On the other hand, substitution of Arg-295 with Lys (R295K) retained high activity, and R295N, R295S, and R295Q exhibited moderate activity. All other substitutions of Arg-295 had no G6Pase activity, suggesting that the role of Arg-295 is to stabilize the protein either by salt bridge or hydrogen-bond formation. Substitution of Gly-222, however, remained functional unless a basic (Arg or Lys), acidic (Asp), or large polar (Gln) residue was introduced, consistent with the hydrophobic requirement of codon 222, which is predicted to be in the fourth membrane-spanning domain. It is possible that Arg-83 is involved in stabilizing the enzyme (His)-phosphate intermediate formed during G6Pase catalysis. There exist 9 conserved His residues in human G6Pase. His-9, His-119, His-252, and His-353 reside on the same side of the endoplasmic reticulum membrane as Arg-83. Whereas H119A mutant G6Pase had no enzymatic activity, H9A, H252A, and H353A mutant G6Pases retained significant activity. Substitution of His-119 with amino acids of diverse structures also yielded mutant G6Pase with no activity, suggesting that His-119 is the phosphate acceptor in G6Pase catalysis. We also present data demonstrating that the carboxyl-terminal 8 residues in human G6Pase are not essential for G6Pase catalysis.

Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B

“…Mutations at the equivalent position of Arg83 (1.08) in human G6Pase (alignment is shown in Fig. S1A) to any other amino acid residue, including Lys, also abolished enzyme activity (21). In particular, mutation of R83C has been directly related to glycogen storage disease type 1a (10,22).…”

“…His163 (2.04) stabilizes the transition state intermediate and catalyzes the cleavage of the terminal PO 4 group from the substrate (18). Mutations at the equivalent position of His119 (2.04) in human G6Pase to any other amino acid residue have been shown to abolish its enzyme activity (21). In ecPgpB, conserved Arg104 (1.08) interacts with the catalytic His207 (3.08) and potentially with the substrate.…”

Identification of a novel phosphatase sequence motif

“…All tested amino acid substitutions for those two residues (including the conservative Lys substitution for Arg 83) resulted in loss of glucose-6-phosphatase activity. Finally, although most of the conserved amino acids of domain 3 are present in the human glucose-6-phosphatase, those residues were not investigated in that study (Lei et al, 1995). The proposed glucose-6-phosphatase topology model predicts domain 3 on the opposite and noncatalytic side of the membrane from domains I and 2.…”

“…In addition, earlier experiments on glucose-6-phosphatase indicated that a phosphoenzyme intermediate is formed during catalysis, and that a histidine residue is the phosphoryl acceptor (Feldman & Butler, 1972;Countaway et al, 1988). Using sitedirected mutagenesis to study the structure-function relationship of the human glucose-6-phosphatase, Lei et al (1995) separately introduced amino acid substitutions for Arg 83 and His I19 (the conserved histidine present in domain 2) and tested the mutated enzymes for glucose-6-phosphatase activity. All tested amino acid substitutions for those two residues (including the conservative Lys substitution for Arg 83) resulted in loss of glucose-6-phosphatase activity.…”

The Molecular Basis of Glycogen Storage Disease Type 1a

“…The eight-amino acid FLAG marker peptide, DYKDDDDK (Scientific Imaging Systems, Eastman Kodak, CT) was used to tag the amino or carboxyl terminus of G6Pase as described previously (18). The two outside PCR primers for G6Pase mutants that contain mutations upstream of the DraIII site are nucleotides 77-96 (G1; sense) and nucleotides 625-602 of G6Pase-DraIII (I2; antisense) (29), and the two outside PCR primers for mutants that contain mutations downstream of the DraIII site are nucleotides 611-634 (I1; sense) and nucleotides 1150 -1133 of G6Pase-DraIII (G2; antisense). The two outside primers for G6Pase R170Q, G184E, G184V, G188D, and G188R mutants are G1 and G2.…”