Publications (5)8.72 Total impact
Chemistry 11/2011; 17(50):13983-6. · 5.93 Impact Factor
Article: Direct observation of a ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor04/2002;
Article: Kinetic properties of HIV-1 protease produced by total chemical synthesis with cysteine residues replaced by isosteric L-α-amino-n-butyric acid[show abstract] [hide abstract]
ABSTRACT: Human immunodeficiency virus-1 protease, produced by total chemical synthesis with the cysteine residues replaced by L--amino-n-butyric acid ([Aba67,95] HIV-1 PR), has been used extensively for the X-ray crystallographic structural analysis of the enzyme and its complexes utilized in drug design. Here we report kinetic studies on the synthetic enzyme. The pH optimum is 5.5 at ionic strengths of 0.1 and 1.0. The acid pH optimum is due to a decrease in binding affinity at higher pH values rather than to a reduction in catalytic efficiency. Activity is markedly increased by high ionic strength, although the major effect is on Km and not Kcat. The effect of pH and ionic strength on the kinetic constants determined for substrates and inhibitors is demonstrated and attention is drawn to the need for assay conditions to be explicitly reported in studies on inhibitor activity. The effect of a number of inhibitors has been measured against the synthetic enzyme and a recombinant HIV-1 PR. This work shows that [Aba67,95] HIV-1 PR has full enzymatic activity and normal kinetic properties.Letters in Peptide Science 08/1995; 2(2):99-107.
Article: Structural engineering of the HIV‐1 protease molecule with a β‐turn mimic of fixed geometry[show abstract] [hide abstract]
ABSTRACT: An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' β-turn mimic of predetermined three-dimensional structure. The mimic β-turn replaced residues Gly16, 17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' β-turns in the native, unliganded enzyme. The β-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16, 17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the β-turn at residues 15–18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid β-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a “molecular kit set” approach to protein design and synthesis.Protein Science 06/1993; 2(7):1085 - 1091. · 2.80 Impact Factor
Article: Ion-spray tandem mass spectrometry in peptide synthesis: Structural characterization of minor by-products in the synthesis of ACP(65–74)[show abstract] [hide abstract]
ABSTRACT: Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65–74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Gly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with for 1 h at 0°C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65–74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.Analytical Biochemistry.