Analysis of ergonomics problems contact-centers

The analysis of the ergonomic problems of the contact center. It allocates main factors of influence on man- operator

Quantitative Analysis of the Effect of Salt Concentration on Enzymatic Catalysis

Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt−rate profiles:  kcat/KM = (kcat/KM)MAX/[1 + ([Na+]/KNa+)n‘], where (kcat/KM)MAX is the physical limit of kcat/KM, KNa+ is the salt concentration at which kcat/KM = (kcat/KM)MAX/2, and −n‘ is the slope of the linear region in a plot of log(kcat/KM) versus log [Na+]. The value of n‘ is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with kcat/KM values that can exceed 109 M-1 s-1. Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7, Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n‘. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n‘. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n‘, as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt−rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis

Catalysis by Ribonuclease A Is Limited by the Rate of Substrate Association^†

The value of kcat/KM for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed 109 M-1 s-1 in a solution of low salt concentration. This value approaches that expected for the diffusional encounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis by RNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis were determined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningful analysis. The pKa values determined from pH−kcat/KM profiles at 0.010, 0.20, and 1.0 M NaCl are inconsistent with the known macroscopic pKa values of RNase A. This incongruity indicates that catalysis of RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. The effect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistent with a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilized by Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolved to become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst

Consequences of the Endogenous <i>N</i>‑Glycosylation of Human Ribonuclease 1

Ribonuclease 1 (RNase 1) is the most prevalent human homologue of the archetypal enzyme RNase A. RNase 1 contains sequons for N-linked glycosylation at Asn34, Asn76, and Asn88 and is N-glycosylated at all three sites in vivo. The effect of N-glycosylation on the structure and function of RNase 1 is unknown. By using an engineered strain of the yeast Pichia pastoris, we installed a heptasaccharide (Man5GlcNAc2) on the side chain of Asn34, Asn76, and Asn88 to produce the authentic triglycosylated form of human RNase 1. As a glutamine residue is not a substrate for cellular oligosaccharyltransferase, we used strategic asparagine-to-glutamine substitutions to produce the three diglycosylated and three monoglycosylated forms of RNase 1. We found that the N-glycosylation of RNase 1 at any position attenuates its catalytic activity but enhances both its thermostability and its resistance to proteolysis. N-Glycosylation at Asn34 generates the most active and stable glycoforms, in accord with its sequon being highly conserved among vertebrate species. These data provide new insight on the biological role of the N-glycosylation of a human secretory enzyme

Stereoelectronic and Steric Effects in the Collagen Triple Helix: Toward a Code for Strand Association

Collagen is the most abundant protein in animals. The protein consists of a helix of three strands, each with sequence X−Y−Gly. Natural collagen is most stable when X is (2S)-proline (Pro) and Y is (2S,4R)-4-hydroxyproline (4R-Hyp). We had shown previously that triple helices in which X is (2S,4S)-4-fluoroproline (4S-Flp) or Y is (2S,4R)-4-fluoroproline (4R-Flp) display hyperstability. This hyperstability arises from stereoelectronic effects that preorganize the main-chain dihedral angles in the conformation found in the triple helix. Here, we report the synthesis of strands containing both 4S-Flp in the X-position and 4R-Flp in the Y-position. We find that these strands do not form a stable triple helix, presumably because of an unfavorable steric interaction between fluoro groups on adjacent strands. Density functional theory calculations indicate that (2S,3S)-3-fluoroproline (3S-Flp), like 4S-Flp, should preorganize the main chain properly for triple-helix formation but without a steric conflict. Synthetic strands containing 3S-Flp in the X-position and 4R-Flp in the Y-position do form a triple helix. This helix is, however, less stable than one with Pro in the X-position, presumably because of an unfavorable inductive effect that diminishes the strength of the interstrand 3S-FlpCO···HNGly hydrogen bond. Thus, other forces can counter the benefits derived from the proper preorganization. Although (Pro−Pro−Gly)7 and (4S-Flp−4R-Flp−Gly)7 do not form stable homotrimeric helices, mixtures of these two peptides form stable heterotrimeric helices containing one (Pro−Pro−Gly)7 strand and two (4S-Flp−4R-Flp−Gly)7 strands. This stoichiometry can be understood by considering the cross sections of the two possible heterotrimeric helices. This unexpected finding portends the development of a “code” for the self-assembly of determinate triple helices from two or three strands

Stereoelectronic Effects on Collagen Stability: The Dichotomy of 4-Fluoroproline Diastereomers

Collagen is the most abundant protein in animals. Natural collagen consists of a triple helix of (Xaa-Yaa-Gly)n chains, in which the Xaa and Yaa residues are often l-proline. Here, a (2S,4S)-4-fluoroproline (flp) residue is shown to be greatly stabilizing in the Xaa position (but destabilizing in the Yaa position). In contrast, a (2S,4R)-4-fluoroproline (Flp) residue is shown to be greatly destabilizing in the Xaa position (but stabilizing in the Yaa position). The dichotomous effect of the diastereomers appears to arise from a gauche effect, which alters pyrrolidine ring pucker and hence properly (or improperly) preorganizes main-chain dihedral angles. Thus, the rational use of stereoelectronic effects can enhance the conformational stability of a protein

Turnover of PIP₃ by PTEN-L in solutions of various salt concentrations.

<p>Reactions were performed in 50 mM Tris–HCl buffer, pH 7.6, containing NaCl (0, 100, or 200 mM), EDTA (2.0 mM), MESG (0.20 mM), and DTBA (40 mM), and were initiated with the addition of PTEN-L to 20 nM. Values are for maximum reaction velocity (± SE) at <10% turnover of substrate in reactions performed in triplicate or more. The resulting kinetic parameters are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116898#pone.0116898.t001" target="_blank">Table 1</a>. </p

Energetics of an <i>n</i> → <i>π</i>* Interaction that Impacts Protein Structure

The trans/cis ratio of the amide bond in N-formylproline phenylesters correlates with electron withdrawal by a para substituent. The slope of the Hammett plot (ρ = 0.26) is indicative of a substantial effect. This effect arises from a favorable n → π* interaction between the amide oxygen and ester carbonyl. In a polypeptide chain, an analogous interaction can stabilize the conformation of trans peptide bonds, α-helices, and polyproline type-II helices

Turnover of PIP₃ by K13A PTEN in solutions of various salt concentrations.

<p>Reactions were performed in 50 mM Tris–HCl buffer, pH 7.6, containing NaCl (0, 100, or 200 mM), EDTA (2.0 mM), MESG (0.20 mM), and DTBA (40 mM), and were initiated with the addition of K13A PTEN to 20 nM. Values are for maximum reaction velocity (± SE) at <10% turnover of substrate in reactions performed in triplicate or more. The resulting kinetic parameters are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116898#pone.0116898.t001" target="_blank">Table 1</a>.</p

Silencing an Inhibitor Unleashes a Cytotoxic Enzyme

The ribonuclease inhibitor (RI) is a cytosolic protein and a potent inhibitor of bovine pancreatic ribonuclease (RNase A). Amphibian homologues and variants of RNase A that evade RI are cytotoxic. Here, we employ RNA interference along with amphibian and mammalian ribonucleases to demonstrate that RI protects cells against exogenous ribonucleases. These data indicate an imperative for the molecular evolution of RI and suggest a means of enhancing the cytotoxicity of mammalian ribonucleases