Structural Biochemistry/Proteins/Total Chemical Synthesis of a D-Enzyme: The Enantiomers of HIV-1 Protease Show Demonstrations of Reciprocal Chiral Substrate Specificity

Purpose
The total chemical synthesis of a D-Enzyme experiment was conducted by R. C. deL. Milton, S.C. F. Milton, and S. B. H. Kent, which found enzyme enantiomers exhibiting reciprocal chiral specificity on peptide sequences. The concept of L-configuration of amino acids predominates in living organisms while the D-configuration remains biologically inactive; Milton et al. examined the ability of enzymes to distinguish and react with a specific enantiomer over the other.

Methods
The following properties of D-HIV PR and L-HIV PR were analyzed: covalent structure, physical properties, circular dichroism spectra, and enzymatic activity. After the total synthesis of D-HIV PR and L-HIV PR, the new synthesized L- and D- sequences of HIV PR were initially protected and then deprotected to allow the folding of their secondary and tertiary structures. The second method used reversed-phase high-performance liquid chromatography which resulted to identical retention rates of the two polypeptide sequences. It was further examined by ion-mass spectroscopy that both polypeptide sequences had the same molecular weight. This method found that both the D-HIV PR and L-HIV PR sequences had the same covalent structure. Despite having the same covalent structure between D-HIV PR and L-HIV PR, differences arise within its chiral features; using a circular ion spectra proved the expected equal but opposite optical activity of the enantiomers. Within a fluorogenic assay containing a hexapeptide analog of a GAG cleavage site was used as a substrate to test the reactivity of the enantiomers. Both enzymes were equally active, yet exhibited reciprocal chiral specificity; reciprocal chiral specificity was apparent when L-enzyme degraded only the L-substrate and D-enzyme degraded only the D-substrate. In addition, reactivity of the D-HIV PR and L-HIV PR were further tested with enantiomers of an inhibitor called MVT101. As expected its corresponding enzyme determined the effectiveness of the inhibitor; L-MVT101 inhibited L-HIV PR but not D-HIV PR, and D-MVT101 inhibited D-HIV PR but not L-HIV PR. The folding of the polypeptide chains into the three-dimensional structure holds importance to the specificity and catalytic activity of HIV-1 protease. D-HIV PR and L-HIV PR displayed mirror images of each other within the secondary, supersecondary, tertiary, and quaternary structure. In the primary structure, only one chiral amino acid was introduced in the synthesis of the polypeptide chain for D-HIV PR and L-HIV PR; the consequence of this one chiral amino acid in the polypeptide backbone resulted to mirror images of the secondary, supersecondary, tertiary, and quaternary structures.

Conclusion
The results of this experiment conclude that the two configurations of the enantiomer are reactive and should be reactive in vivo, yet due to evolution the L-proteins are prevalent in living organisms while D-proteins are biologically inactive.