Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease

• Published in: Biochemistry (Easton) Vol. 31; no. 42; pp. 10153 - 10168
• Main Authors: Tomaszek, Thaddeus A, Moore, Michael L, Strickler, James E, Sanchez, Robert L, Dixon, J. Scott, Metcalf, Brian W, Hassell, Anne, Dreyer, Geoffrey B, Brooks, Isobel
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 27.10.1992
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HIV Protease - chemistry; HIV Protease - metabolism; HIV-1 - enzymology; human immunodeficiency virus 1; Hydrolases; Indicators and Reagents; Kinetics; L-Lactate Dehydrogenase - metabolism; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Muscles - enzymology; Myocardium - enzymology; Oligopeptides - chemical synthesis; Oligopeptides - chemistry; Oligopeptides - metabolism; Peptide Fragments - metabolism; Protein Conformation; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity; Swine; X-Ray Diffraction; HIV-1 virus; Enzyme; Cleavage site; Retroviridae; Environmental factor; Lactate dehydrogenase; In vitro; Virus; Aspartic proteinase; Enzymatic activity; Substrate specificity; pH; Lentivirinae; Human immunodeficiency virus; Recognition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00157a003

The muscle and heart lactate dehydrogenase (LDHs) of rabbit and pig are specifically cleaved at a single position by HIV-1 protease, resulting in the conversion of 36-kDa subunits of the oligomeric enzymes into 21- and 15-kDa protein bands as analyzed by SDS-PAGE. While the proteolysis was observed at neutral pH, it became more pronounced at pH 6.0 and 5.0. The time courses of the cleavage of the 36-kDa subunits were commensurate with the time-dependent loss of both quaternary structure and enzymatic activity. These results demonstrated that deoligomerization of rabbit muscle LDH at acidic pH rendered its subunits more susceptible to proteolysis, suggesting that a partially denatured form of the enzyme was the actual substrate. Proteolytic cleavage of the rabbit muscle enzyme occurred at a decapeptide sequence, His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp (scissile bond denoted throughout by an asterisk), which constitutes a "strand-loop" element in the muscle and heart LDH structures and contains the active site histidyl residue His-193. The kinetic parameters Km, Vmax/KmEt, and Vmax/Et for rabbit muscle LDH and the synthetic decapeptide Ac-His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp-NH2 were nearly identical, suggesting that the decapeptide within the protein substrate is conformationally mobile, as would be expected for the peptide substrate in solution. Insertion of part of this decapeptide sequence into bacterial galactokinase likewise rendered this protein susceptible to proteolysis by HIV-1 protease, and site-directed mutagenesis of this peptide in galactokinase revealed that the Glu residue at the P2' was important to binding to HIV-1 protease. Crystallographic analysis of HIV-1 protease complexed with a tight-binding peptide analogue inhibitor derived from this decapeptide sequence revealed that the "strand-loop" structure of the protein substrate must adopt a beta-sheet structure upon binding to the protease. The Glu residue in the P2' position of the inhibitor likely forms hydrogen-bonding interactions with both the alpha-amide and gamma-carboxylic groups of Asp-30 in the substrate binding site.