Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.
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PMID:Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. 38 11

The Bacillus subtilis gene (pyrB), which encodes aspartate transcarbamylase (ATCase), was cloned on a HindIII restriction endonuclease fragment inserted into the pUC13 plasmid vector. B. subtilis pyrB was expressed in Escherichia coli, as judged by complementation of E. coli pyrB mutants and production of enzyme that was specifically inhibited by antibody directed against B. subtilis ATCase. The extent of expression was strongly dependent on the orientation of the inserted DNA in the vector, which suggested that transcription was initiated from vector-borne (rather than B. subtilis) promoters. The entire 1098-base pair HindIII fragment of B. subtilis DNA was sequenced by the Maxam-Gilbert method. The amino acid sequence of B. subtilis ATCase was deduced from a 305-codon open reading frame and agreed very well with analyses of the purified enzyme. Comparison of the sequence of B. subtilis ATCase with that of E. coli ATCase catalytic subunit, for which the three-dimensional structure is known, revealed many homologous residues of probable importance in catalysis and structural folding of ATCases. The significance of homology to E. coli ornithine transcarbamylases was also analyzed. The sequences of the 5' and 3' flanking regions to pyrB encode open reading frames in both cases which overlap with pyrB by eight and six codons, respectively. It is probable that these open reading frames encode other enzymes of a coordinately regulated unit. The sequence 5' to pyrB also encodes an mRNA bearing a pyrimidine-rich sequence followed by a typical sequence for a rho-independent transcription terminator. The presence of these elements and the 5' open reading frame suggest that B. subtilis pyrB, like E. coli pyrBI, is regulated by an attenuation mechanism.
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PMID:Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). 301 59

In this article we focus on presenting a broad range of examples illustrating low-energy transitions via hinge-bending motions. The examples are divided according to the type of hinge-bending involved; namely, motions involving fragments of the protein chains, hinge-bending motions involving protein domains, and hinge-bending motions between the covalently unconnected subunits. We further make a distinction between allosterically and nonallosterically regulated proteins. These transitions are discussed within the general framework of folding and binding funnels. We propose that the conformers manifesting such swiveling motions are not the outcome of "induced fit" binding mechanism; instead, molecules exist in an ensemble of conformations that are in equilibrium in solution. These ensembles, which populate the bottoms of the funnels, a priori contain both the "open" and the "closed" conformational isomers. Furthermore, we argue that there are no fundamental differences among the physical principles behind the folding and binding funnels. Hence, there is no basic difference between funnels depicting ensembles of conformers of single molecules with fragment, or domain motions, as compared to subunits in multimeric quaternary structures, also showing such conformational transitions. The difference relates only to the size and complexity of the system. The larger the system, the more complex its corresponding fused funnel(s). In particular, funnels associated with allosterically regulated proteins are expected to be more complicated, because allostery is frequently involved with movements between subunits, and consequently is often observed in multichain and multimolecular complexes. This review centers on the critical role played by flexibility and conformational fluctuations in enzyme activity. Internal motions that extend over different time scales and with different amplitudes are known to be essential for the catalytic cycle. The conformational change observed in enzyme-substrate complexes as compared to the unbound enzyme state, and in particular the hinge-bending motions observed in enzymes with two domains, have a substantial effect on the enzymatic catalytic activity. The examples we review span the lipolytic enzymes that are particularly interesting, owing to their activation at the water-oil interface; an allosterically controlled dehydrogenase (lactate dehydrogenase); a DNA methyltransferase, with a covalently-bound intermediate; large-scale flexible loop motions in a glycolytic enzyme (TIM); domain motion in PGK, an enzyme which is essential in most cells, both for ATP generation in aerobes and for fermentation in anaerobes; adenylate kinase, showing large conformational changes, owing to their need to shield their catalytic centers from water; a calcium-binding protein (calmodulin), involved in a wide range of cellular calcium-dependent signaling; diphtheria toxin, whose large domain motion has been shown to yield "domain swapping;" the hexameric glutamate dehydrogenase, which has been studied both in a thermophile and in a mesophile; an allosteric enzyme, showing subunit motion between the R and the T states (aspartate transcarbamoylase), and the historically well-studied lac repressor. Nonallosteric subunit transitions are also addressed, with some examples (aspartate receptor and BamHI endonuclease). Hence, using this enzyme-catalysis-centered discussion, we address energy funnel landscapes of large-scale conformational transitions, rather than the faster, quasi-harmonic, thermal fluctuations.
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PMID:Folding funnels and conformational transitions via hinge-bending motions. 1059 56