Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.4.6 (urease)
 7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.
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PMID:Kinetic and structural characterization of urease active site variants. 1091 64

Series of liquid photopolymerizable compositions (LPhPC) based on oligouretanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemetacrylate (OCM-2), monomethacrylic ether of ethylene glycol and vinylpyrrolidone (VP) were tested. It was shown that the LPhPC, which contained VP (as basic hydrophylic matrix), OCM-2 (cross-linking agent) and OUM-2000T (to increase adsorption of polymer) was the most optimal. The blend contained 3 g/100 ml of enzyme. ISFET based biosensors for analysis of glucose and urea had the following characteristics: linear response in the range of concentrations 0.1-10 mmol/l, 0.05-20 mmol/l, angle of slope of concentration curve--30 mV/pC, 38 mV/pC, and response time of approximately 10-15, 5-10 min, correspondingly. The value of Km for immobilized urease and beta-glucose oxidase (GOD) achieved 0.85 and 3.1 mmol/l, respectively. It was established that under immobilization conditions at 20 degrees C the residual activity of GOD was about 35% from the initial level, the residual activity of horseradish peroxidase (HRP) and urease was 42% and 20%, respectively. In case of an immobilization of GOD at -50 degrees C its residual activity reached almost 50% from the initial level. It was investigated how different sources of UV radiation and different substances (including specific and non-specific substrates) influenced stability of the enzymes in the LPhPC and in the prepared membrane at storage. Dynamics of changes of enzyme activity at the process of photo immobilization was characterized, and requirements for enzyme maximal storage were selected. The proposed LPhPC may be prepared in advance since enzymes do not lose their activity during 2 months. Therefore, two processes, i.e. manufacturing of a transducer and preparation of a biological membrane on its surface, can be combined in one. In order to achieve this, approaches of modern electronics, such as for example photolithography, can be used. The developed LPhPC is homogenous, non-active to biological substances, permeable for the analyzed sample, can be prepared using a simple immobilization procedure, and has a defined hydrophobic-hydrophilic balance and sufficient level of adhesion to transducer surfaces. These all cover the requirements to modern biosensors.
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PMID:Liquid photopolymerizable compositions as immobilized matrix of biosensors. 1738 43