The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 Å. Arginine and asparagine could provide only one hydrogen bond of 2.52 and 3.02 Å, respectively. To test this model, we purified recombinant human Gln 188 -, Arg 188 -, and Asn 188 -GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln 188 -GALT displaced 80 ؎ 7.0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg 188 -and Asn 188 -GALT released more glu-1-P (170 ؎ 8.0 and 129 ؎ 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln 188 -GALT produced 80,030 ؎ 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg 188 -and Asn 188 -GALT released only 600 ؎ 71.2 and 2960 ؎ 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal.The enzyme galactose-1-phosphate uridyltransferase (GALT) 1 (EC 2.7.7.12) catalyzes the conversion of UDP-glucose (UDP-Glu) and galactose-1-phosphate (gal-1-P) to form glucose-1-phosphate (glu-1-P) and UDP-galactose (UDP-Gal) in the evolutionarily conserved Leloir pathway of galactose metabolism (1). The enzymology of GALT has been intensively studied using GALT purified from Escherichia coli (2, 3). Both E. coli and human GALT enzymes catalyze the conversion of UDP-Glu and gal-1-P to UDP-Gal and glu-1-P via a double displacement mechanism (2-4) (Fig. 1). Under normal physiological conditions, UDP-Glu binds to GALT to form a GALT-UDP-Glu intermediate. Glu-1-P is subsequently released, whereas the GALT enzyme remains bound to UMP. This GALT-UMP intermediate has been isolated and crystallized (5-8). Gal-1-P then reacts with the GALT-UMP complex to form UDP-Gal, freeing the GALT enzyme for continued catalysis. GALT does not use nucleoside di-or...
Sixty-six students rated written jokes on funniness and on eight other scales. Jokes were varied on the equity of retaliation in an exchange of insults, the relative status of the two parties in this exchange, and in another set of jokes, the popularity of a famous victim of the joke squelch. Overretaliation decreased rated funniness and rated resolution ("sensicalness") of the joke. Control for resolution with analysis of covariance reduced the effect of degree of retaliation on funniness to nonsignificance. Jokes with disliked victims were rated as both funnier and higher in resolution than were jokes with neutral or liked victims, but it was necessary to control for both resolution and feltfreedom ratings to eliminate the effect on funniness. Incongruity (surprise) ratings were not affected by any treatment. Results are interpreted as supporting a proposed integration of disparagement theories with incongruityresolution theories of humor.
A Center for Substance Abuse Treatment Knowledge Application Program based on cognitive-behavioral and self-management treatment approaches and targeted to older adults with substance abuse was provided through a community behavioral health center. A sample of 199 adults aged 50 and above participated in the 18-session program. Observations were made at intake and 6 months after intake. Program completers versus noncompleters differed significantly over time, favoring completers with regard to decreased use of nonmedical prescription drugs, improved cognitive functioning, improved mental health, increased vitality, and lack of bodily pain. Significant time effects were noted in participants' decreased use of alcohol and binge drinking, reduced stress, fewer emotional problems, a decrease in having to reduce important activities, and increased prescription of medication for psychological problems. Participants also reported significant improvement in their social functioning, and their physical health and emotional problems had less impact on what they were able to do.
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