ARGININE TO TRYPTOPHAN SUBSTITUTION IN THE ACTIVE-SITE OF A HUMAN LACTATE-DEHYDROGENASE VARIANT - LDHB (ASTERISK) GUA1 - POSTULATED EFFECTSON SUBUNIT STRUCTURE AND CATALYSIS

A variant of lactate dehydrogenase (LDHBGUA1) was previously identifi ed among the Guaymi Indians of Panama and Costa Rica. The LDHBGUA1 va riant is enzymatically inactive; however, the variant subunits alter t he electrophoretic mobility of the tetramers that include active LDHA and LDHB subunits. The kinetic properties of the tetrameric enzyme, co mprised of inactive B plus active A subunits, are similar to propertie s of the heterotetramers with active B subunits, except for the reduce d specific activity. We have determined that a single C . G to T . A t ransition changes an Arg to a Trp at amino acid residue 106. This subs titution explains the increase;in net negative charge observed by prot ein electrophoresis. This Arg 106 residue is absolutely conserved thro ughout evolution. Published high-resolution crystal structures of LDH reveal that this residue is within the hinge of a loop that closes ove r the active site of the subunit upon binding of substrate and cofacto r and also has a direct role in catalysis. Computer modeling of the va riant 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 acti vity through the inability of the uncharged tryptophan side-chain to p olarize the substrate carbonyl bond. This would explain the loss of ca talytic function with maintenance of normal kinetic properties in the heterotetramers containing the variant subunits. The ability to mainta in normal, tissue-specific kinetic properties could explain the absenc e of clinical manifestations in the homozygous LDHBGUA1 individuals.