Random mutant libraries with substitutions at the interface between the N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochrome c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the libraries. Mutants with these combinations are biologically functional. Enthalpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation (delta GD) for the ferri form of the wild-type protein and 13 interface variants. Changes in delta GD cannot be allocated solely to enthalpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of delta GD values for single versus multiple amino acid substitutions indicates that the helices interact thermodynamically. Changes in delta GD are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of delta GD values for the interface variants and nearly 90 non-cytochrome c variants to side-chain model data leads to several conclusions. First, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently. Second, despite octanol being a poor model for protein interiors, octanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unlike cyclohexane and octanol, the Dayhoff mutation matrix predicts stability changes for a variety of substitutions, even at interacting sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Induced fit has been postulated to be an important component of ligand interactions with proteins, including protein-DNA interactions. We imagined that the entropic cost of induced fit might be highly dependent on the local protein sequence context around critical contact residues. To investigate this question, we analyzed the basis for active or inactive phenotypes found in a library of combinatorial sequence variants of a surface-located helix-loop peptide which is essential for the anticodon-binding activity of a class I tRNA synthetase. Molecular dynamics simulations of the domain encompassing the helix-loop peptide of the active variants consistently demonstrated fixation of the local motion of five critical (for function) residues which are highly mobile in inactive variants. Additional experiments with other rationally chosen mutants extended the correlation between phenotype and motion of these vital residues. We propose that the need for fixation of local motion is an important constraint on sequences of surface peptides which form parts of RNA-binding sites. The fixation of motion of critical residues in the unbound protein can significantly reduce the entropic cost of complex formation by induced fit.

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormones are potent, instantaneous, and reversible inhibitors of ethanol oxidation catalyzed by isozymes of class I and II human alcohol dehydrogenase (ADH). None of the thyroid hormones inhibits class III ADH. At pH 7.40 the apparent Ki values vary between 55 and 110 microM for triiodothyronine, 35 and greater than 200 microM for thyroxine, and 10 and 23 microM for triiodothyroacetic acid. The inhibition is of a mixed type toward both NAD+ and ethanol. The binding of the thyroid hormone triiodothyronine to beta 1 gamma 1 ADH is mutually exclusive with 1,10-phenanthroline, 4-methylpyrazole, and testosterone, identifying a binding site(s) for the thyroid hormones, which overlap(s) both the 1,10-phenanthroline site near the active site zinc atom and the testosterone binding site, the latter being a regulatory site on the gamma-subunit-containing isozymes and distinct from their catalytic site. The inhibition by thyroid hormones may have implications for regulation of ADH catalysis of ethanol and alcohols in the intermediary metabolism of dopamine, norepinephrine, and serotonin and in steroid metabolism. In concert with other hormonal regulators, e.g., testosterone, the rate of ADH catalysis is capable of being fine tuned in accord with both substrate and modulator concentrations.

Cu(II)-substituted carboxypeptidase A catalyzes the hydrolysis of oligopeptides and their depsipeptide (ester) analogues. Stopped-flow fluorescence assays demonstrate that relative to the zinc enzyme the Cu enzyme can have kcat/Km values up to 24% toward esters but only up to 2.5% toward the corresponding peptides. Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II) electron paramagnetic resonance spectra. Low concentrations of 1,10-phenanthroline (OP) (0.1-2.5 microM) markedly increase activity toward furanacryloyl-Phe-Phe (up to 8% of the zinc enzyme), but higher concentrations inhibit, resulting in complete inhibition at 0.8 mM OP. The non-metal-binding, hydrophobic analogues m- and p-phenanthroline are only activators of peptide hydrolysis, even at 1 mM. Activation is likely due to a modifier binding to a hydrophobic locus and either displacing an inhibitory peptide binding mode or inducing a conformational change in the active site.