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At least 613 records · Page 34Linked to original sources
PubMed1991

Linear free energy relationships in enzyme binding interactions studied by protein engineering.

Experiments on mutants of tyrosyl-tRNA synthetase have shown that there can be linear free energy relationships (LFERs) between changes in activation free energies and changes in binding energies when groups are deleted that bind to non-reacting parts of the substrate (Fersht et al., 1986, 1987). It has now been proposed (Straub and Karplus, 1990) that such LFERs can occur for the mutation of hydrogen bonding groups only for the limiting examples of Brønsted beta of 0, 1 or infinity, and that fractional values of beta are not permissible. The reasoning behind this is that the energy of a hydrogen bond is not linear with distance and the (false) premise that an LFER requires that there is a linear relationship between bond energy and distance. We show from a simple model how LFERs can arise for binding interactions and how they can give fractional values of beta, in accord with experimental evidence. An LFER occurs between binding and catalysis when a set of interactions exists in which each member contributes to the binding energy of the transition state the same fraction of the binding energy it contributes to the products (both relative to the ground state).

Binding, Competitive↗
PubMed1994

Protein engineering of the relative specificity of glucoamylase from Aspergillus awamori based on sequence similarities between starch-degrading enzymes.

Aspergillus glucoamylase catalyzes hydrolysis of D-glucose from non-reducing ends of starch with an approximately 300-fold (kcat/Km) preference for the alpha-1,4- over the alpha-1,6-glucosidic linkage determined using the substrates maltose and isomaltose. It is postulated that as most amylolytic enzymes act on either the alpha-1,4- or alpha-1,6-linkages, sequence comparison between active-site regions should enable the correlation of the substrate bond specificity with particular residues at key positions. Therefore, the already high bond-type selectivity in Aspergillus glucoamylase could theoretically be augmented further by three single mutations, Ser119-->Tyr, Gly183-->Lys and Ser184-->His, in two separate active-site regions. These mutants all had slight increases in activity as compared with the wild-type enzyme towards the alpha-1,4-linked maltose; this was due to lower Km values as well as small decreases in activity towards isomaltose. This latter decrease in activity was a result of higher Km values and a decrease in kcat for the Ser184-->His mutant. As a consequence, the selectivity of the three glucoamylase mutants for alpha-1,4- over alpha-1,6-linked disaccharides is enhanced 2.3- to 3.5-fold. In addition, the introduction of a cationic side chain in Gly183-->Lys and Ser184-->His glucoamylase, broadens the optimal pH range for activity towards acidic as well as alkaline conditions.

Amino Acid Sequence↗
PubMed2001

Genetic and protein engineering of diagnostic enzymes, cholesterol oxidase and xylitol oxidase.

For a long time, clinical diagnosis has been made mainly using chemical methods. Recently, several excellent substrate-specific enzymes have been developed and these enzymes are used as diagnostic catalysts. Using enzymes, it is possible to assay for a specific substance from specimens of serum or urine without the need for isolation of the substance which simplifies the process and shortens the assay time. Furthermore, the use of enzymatic assay methods for diagnosis has been facilitated by the developments in genetic engineering which made it possible to overproduce enzymes inexpensively. Here, we review the diagnostic enzymes, cholesterol oxidase and xylitol oxidase, which were successfully overproduced in our laboratory. In particular, the catalytic activity and pH and thermal stabilities of cholesterol oxidase were improved.

Journal Article↗
PubMed1988

Protein engineering in studies on metabolism of toxic substances.

Expression of cytochrome P-450s, drug metabolizing enzymes, from their coding sequences in heterologous cells were developed and the enzymatically active P-450s were synthesized. cDNAs of various chimera or mutant P-450s were constructed and expressed. Thus, regions or residues important for the function of cytochrome P-450 were analyzed.

Animals↗
PubMed1996

Protein Engineering of Insulin: [B9Glu, B10Asp] Human Insulin.

B9Ser and B10His of the insulin B chain are substituted respectively by Glu and Asp using a gapped duplex DNA approach for site-directed mutagenesis. A mutant insulin-[B9Glu, B10Asp] human insulin was obtained. The receptor binding capacity of the mutant insulin is 34.4% as that of porcine insulin. However, the in vivo biological activity of [B9Glu, B10Asp] human insulin is almost as the same as that of porcine insulin.

Journal Article↗
PubMed1996

Protein Engineering of Insulin: [B9 Glutamic Acid] Human Insulin.

B9Ser of insulin B chain was substituted by Glu using site-directed mutagenesis to obtain a fast-acting insulin-[B9Glu] human insulin. The receptor binding capacity and in vivo biological activity of [B9Glu] human insulin are 21% and 40% as those of porcine insulin respectively.

Journal Article↗
PubMed2000

Engineering therapeutic proteins.

Many early drug candidates derived from biotechnology failed in clinical trials because of their low affinity/specificity, short half-lives or immunogenicity. Protein engineering techniques have been applied to circumvent some of the problems that hindered these earlier trials, resulting in clinical benefits from a range of engineered antibodies and other proteins.

Animals↗
PubMed2004

The use of recombinant methods and molecular engineering in protein crystallization.

Recombinant techniques are routinely used for the preparation of protein samples for structural studies including X-ray crystallography. Among other benefits, these methods allow for a vast increase in the amount of obtained protein as compared to purification from source tissues, ease of purification when fusion proteins containing affinity tags are used, introduction of SeMet for phasing, and the opportunity to modify the protein to enhance its crystallizability. Protein engineering may involve removal of flexible regions including termini and interior loops, as well as replacement of residues that affect solubility. Moreover, modification of the protein surface to induce crystal growth may include rational engineering of surface patches that can readily mediate crystal contacts. The latter approach can be used to obtain proteins of crystals recalcitrant to crystallization or to obtain well-diffracting crystals in lieu of wild-type crystals yielding data to limited resolution. This review discusses recent advances in the field and describes a number of examples of diverse protein engineering techniques used in crystallographic investigations.

Chemistry Techniques, Analytical↗
PubMed1995

Engineering membrane proteins.

Much of the research on integral membrane proteins mirrors that on soluble proteins; however, membrane protein engineering also has its own ends and means, many of which take advantage of the peculiar situation of membrane proteins, whose chains are distributed between one lipidic and two aqueous phases. Extramembrane loops have been shortened, cut, or elongated with segments forming proteolytic cleavage sites, foreign epitopes, extra transmembrane segments, or even whole proteins, with the aim of facilitating purification, biochemical/biophysical studies, or crystallogenesis. Transmembrane alpha-helices have been deleted, duplicated, exchanged, transported into a foreign context or replaced with synthetic peptides, in order to both understand their integration into, and assembly in, the membrane and unravel their functional role. Insertion of cysteine residues has been the basis for a great diversity of experiments, ranging from the exploration of secondary, tertiary and quaternary structures of the transmembrane region to the creation of anchoring points for reporter molecules. Chemical engineering--the synthesis of protein fragments or even of whole proteins--offers particularly exciting new prospects, given the small size of folding domains in alpha-helical membrane proteins. Membrane protein engineering is rapidly developing its own agenda of questions and tool chest of techniques.

Bacteriorhodopsins↗