Arginine to tryptophan substitution in the active site of a human lactate dehydrogenase variant--LDHB GUA1: postulated effects on subunit structure and catalysis.
A variant of lactate dehydrogenase (LDHB GUA1) was previously identified among the Guaymi Indians of Panama and Costa Rica. The LDHB GUA1 variant is enzymatically inactive; however, the variant subunits alter the electrophoretic mobility of the tetramers that include active LDHA and LDHB subunits. The kinetic properties of the tetrameric enzyme, comprised of the inactive B plus active A subunits, are similar to properties of the heterotetramers with active B subunits, except for the reduced specific activity. We have determined that a single C.G to T.A transition changes an Arg to a Trp at amino acid residue 106. This substitution explains the increase in net negative charge observed by protein electrophoresis. This Arg 106 residue is absolutely conserved throughout evolution. Published high-resolution crystal structures of LDH reveal that this residue is within the hinge of a loop that closes over the active site of the subunit upon binding of substrate and cofactor and also has a direct role in catalysis. Computer modeling of the variant enzyme suggests that replacement of this Arg residue with a Trp does not induce significant change in the structure of the active site. However, this substitution would result in disruption of enzyme activity through the inability of the uncharged tryptophan side-chain to polarize the substrate carbonyl bond. This would explain the loss of the catalytic function with maintenance of normal kinetic properties in the heterotetramers containing the variant subunits. The ability to maintain normal, tissue-specific kinetic properties could explain the absence of clinical manifestations in the homozygous LDHB GUA1 individuals.
Shonnard
G C
GC
Biology and Biotechonology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
Hud
N V
NV
Mohrenweiser
H W
HW
D000483:Alleles; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D003364:Costa Rica; D018360:Crystallography, X-Ray; D017931:DNA Primers; D014644:Genetic Variation; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010176:Panama; D017354:Point Mutation / Q000235:genetics*; D011487:Protein Conformation; D017421:Sequence Analysis; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D014364:Tryptophan; D001120:Arginine; D007770:L-Lactate Dehydrogenase