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Substrate Access (substrate + access)

Distribution by Scientific Domains

Chemistry	80%

Selected Abstracts

Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain

FEBS JOURNAL, Issue 18 2002
Yoko Kobayashi
Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations , a closed, inactive form and an open, active form , differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure,function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a ,head-to-tail' orientation, two inactive LPL mutants , a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) , were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other. [source]

Effect of substrate size on immunoinhibition of amylase activity,

JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 2 2001
Ilka Warshawsky
Abstract Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10‐fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one. J. Clin. Lab. Anal. 15:64–70, 2001. [source]

Mutations of key hydrophobic surface residues of 11,-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

PROTEIN SCIENCE, Issue 7 2009
Alexander J. Lawson
Abstract 11,-Hydroxysteroid dehydrogenase type 1 (11,-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11,-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered Km values for steroids and an unaltered or increased kcat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild-type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme. [source]

Activation Function of Chloroperoxidase in the Presence of Metal Ions at Elevated Temperature from 25 to 55°C

CHINESE JOURNAL OF CHEMISTRY, Issue 7 2009
Qiang GAO
Abstract The investigation and comparison of chlorination activity of chloroperoxidase (CPO) from Caldariomyces fumago in metal ion solutions to those in pure buffer indicated that CPO could be effectively activated by some alkaline-earth metals and transition metals. The obtained maximum relative activity of CPO was 1.33 time at 75 µmol·L,1 Ca2+, 1.37 time at 90 µmol·L,1 Mg2+, 1.34 time at 90 µmol·L,1 Ni2+, and 1.27 time at 105 µmol·L,1 Co2+ at 25°C. Moreover, the CPO stability against temperature was improved in the presence of the above metal ions. At 55°C, CPO could retain only about 40% of activity whereas 75% and 81% of activity were maintained in Mg2+ and Ca2+ media, respectively. It was suggested that the metal ions bind to the acid-base catalytic groups Glu183, His105 and Asp106 around the active site of CPO, and activate CPO by both an enrichment of substrate concentration and the conformational change of CPO, which are favorable to the substrate access. The analysis of kinetic parameters indicated that the activation was mainly due to an increase in kcat values. The affinity and specificity of CPO to substrates were also improved in these metal ion media. The results in this work are promising in view of industrial applications of this versatile biological catalyst. [source]