Sequence and Structural Features of Enzymes and their Active Sites by EC Class

We have analysed a non-redundant set of 294 enzymes for differences in sequence and structural features between the six main Enzyme Commission (EC) classification groups. This systematic study of enzymes, and their active sites in particular, aims to increase understanding of how the structure of an enzyme relates to its functional role. Many features showed significant differences between the EC classes, including active-site polarity, enzyme size and active-site amino acid propensities. Many attributes correlate with each other to form clusters of related features from which we chose representative features for further analysis. Oxidoreductases have more non-polar active sites, which can be attributed to cofactor binding and a preference for Glu over Asp in active sites in comparison to the other classes. Lyases form a significantly higher proportion of oligomers than any other class, whilst the hydrolases form the largest proportion of monomers. These features were then used in a prediction model that classified each enzyme into its top EC class with an accuracy of 33.1%, which is an increase of 16.4% over random classification. Understanding the link between structure and function is critical to improving enzyme design and the prediction of protein function from structure without transfer of annotation from alignments.     

GROWTH PATTERNS IN NUTATING AND NONNUTATING SUNFLOWER (HELIANTHUS ANNUUS) HYPOCOTYLS

Dark-grown sunflower (Helianthus annuus L.) seedling hypocotyls (15–30 mm) were marked with two rows of lanolin-coated resin beads, and the events of the following 24 hr, in physiological darkness, were recorded on time-lapse video. Nutational movement of the hypocotyl, followed for 20–24 hr for each of 21 seedlings, was found to have a mean period of 153 ± 26 min (ca. 24 C). Displacement of each bead, with time, was measured with a microcomputer-controlled video analyzer, and relative elemental elongation rate and relative growth rate analyses were carried out to determine the spatial and temporal distribution of growth. Relative elemental elongation rates were plotted against distance and time to produce “growth landscapes.” A strongly nutating seedling showed periodic fluctuations in local growth rates that alternated between values of 0.0 hr^−-1 and >0.12 hr^−-1 near the hypocotyl hook. Nearer the base, maximum growth rates were lower but local periodic changes still were evident. Seedlings, in which nutation appeared during the time period analyzed, showed non-synchronous pulses of growth along the axis. With nutational development, these local growth fluctuations became synchronized along each side and phased (usually 180° out of phase) with the coordinated growth fluctuations along the opposite side. In some seedlings the changes from low to high local growth rates occur nearly simultaneously over two-thirds of the active region. In others, basipetally traveling waves of growth are suggested by the growth landscapes.     

Characterization of the oligosaccharides of prolyl hydroxylase, a microsomal glycoprotein

Prolyl hydroxylase is a tetrameric glycoprotein that catalyzes a vital posttranslational modification in the biosynthesis of collagen. The enzyme purified from whole chick embryos (WCE) possesses two nonidentical subunits, alpha and beta, and has been shown by several techniques to reside in the endoplasmic reticulum of chick embryo fibroblasts. The studies described here demonstrate that the larger of the two subunits (alpha) exists in two forms in chick embryo fibroblasts (CEF); these two forms differ in carbohydrate content. The larger alpha subunit, alpha', contains two N-linked high mannose oligosaccharides, each containing eight mannose units; the smaller subunit, alpha, contains a single seven-mannose N-linked oligosaccharide. Both oligosaccharides could be cleaved by endo-beta-N-acetylglucosaminidase H and completely digested with alpha-mannosidase to yield mannosyl-N-acetylglucosamine.     

beta-Chloro-L-alanine inhibition of the Escherichia coli alanine-valine transaminase.

beta-Chloro-L-alanine, an amino acid analog which inhibits a number of enzymes, reversibly inhibited the Escherichia coli K-12 alanine-valine transaminase, transaminase C. This inhibition, along with the inhibition of transaminase B, accounted for the isoleucine-plus-valine requirement of E. coli in the presence of beta-chloro-L-alanine.     

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.     

Mutagenesis of dimeric plasmids by the transposon gamma delta (Tn1000).

The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon gamma delta (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single gamma delta insertion, the occasional recovery of transposon-free plasmids after conjugal transfer has led to alternative hypotheses for F mobilization. We show here that gamma delta-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec+.     

Communication between the corpus cavernosum penis and the corpus spongiosum penis in a bull diagnosed by modified contrast cavernosography: A case report

Traditional methods of penile cavernosography failed to reveal a vascular defect or shunt from the corpus cavernosum penis of a 6-yr-old Brahman bull with a history of acquired failure of penile erection. When a tourniquet was applied to the penis and the contrast medium was introduced into the corpus cavernosum penis under pressure, however, a communication between the corpus cavernosum penis and corpus spongiosum penis was demonstrated. In a normal bull, cavernosography with the introduction of contrast medium under pressure produced a normal radiographic appearance. It is suggested that modification of accepted techniques of cavernosography to incorporate injection of contrast medium under pressure is useful in diagnosing this particular type of vascular abnormality, and may also aid in the detection of smaller vascular shunts from the corpus cavernosum penis to the superficial penile vasculature.     

Purification of human liver fumarylacetoacetase using immunoaffinity chromatography

A method is described to purify fumarylacetoacetase from crude human liver extracts using immunoaffinity chromatography. Immobilized rabbit antibodies specific for beef liver fumarylacetoacetase were used as an immunoadsorbent. With this rapid and specific procedure human liver fumarylacetoacetase could be purified to apparent homogeneity. The molecular weight of native human liver fumarylacetoacetase is approximately 83000 as estimated by gel filtration. The two subunits have a molecular weight of approximately 41000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified human liver fumarylacetoacetase has a broad pH optimum with a maximum at pH 7.2 and a K_m = 2.1 μM towards fumarylacetoacetate.