DNA POLYMORPHIC FORMS OF HUMAN SERUM CHOLINESTERASE

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Human serum cholinesterase, (i.e. butyrylcholinesterase, BChE) is of pharmacological interest because deficiencies (or high levels) of its activity or altered affinities in several variant forms of this enzyme cause an exaggerated response (or resistance) to the muscle-relaxant drug, succinylcholine. Our laboratory has been studying BChE for over 25 years. Dr. Lockridge determined the complete amino acid sequence of purified human serum BChE, and we cloned the gene from a human brain cDNA library, sequenced the entire coding region, and used genomic DNA to determined the size and sequence of the intron regions adjacent to the 4 exons. We found the structural basis of the most common BChE variant; the atypical, or dibucaine-resistant variant, to be a point mutation at nucleotide base 209 (GAT yields GGT). This changes amino acid 70 from Asp yields Gly. The identical mutation has been found, so far, in every person tested with atypical BChE. Since then, we have used PCR amplification to prepare genomic DNA for detailed sequence analyses, and have identified, to date, the structural basis for an additional 22 natural mutations of human BCHE. These include both qualitative alterations in the enzyme, such as fluoride-resistance, an quantitative reductions of one-third (K), two-thirds (J), 90% (H), and essentially no enzymatic activity (silent). The silent group includes point mutations, frame shift mutations, production of unstable enzymes, and enzymes that are not released from liver into the blood. The large number of variants that can now be diagnosed using DNA analyses has required that we use of a DNA structural basis for classifying BCHE genotypes in the future. We are developing new screening methods that should simplify efforts in making surveys for any of these variants in sample populations representing different ethnic groups, and different geographic areas to learn more about the distribution of these variant alleles. Recent advances in our knowledge about the 3-dimensional structure of human BChE make it possible to visualize the exact location and predict the importance of the different amino acid residues. We can identify those key components making up the active center of the esterase. This series of natural BChE variants should be very useful in future studies on how particular modifications in the enzymatic structure can alter the substrate specificity, (particularly with other drugs, such as mivacurium), the response to inhibitors, and how these structural modifications affect the kinetics of the esterase. These studies of the molecular structure and catalytic function of human BChE will improve our understanding of these events at a molecular level for this model pharmacogenetic condition.