The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.

No abstract

The burden of constipation from the patient's perspective has been well described. The aim of this study was to evaluate the cost of managing constipation in patients taking opioids in a specialist palliative care inpatient unit. A retrospective review of the medical records of 58 patients (70 admissions) who died during a six-month period was undertaken to identify prescribing patterns for opioids and oral laxatives and tasks associated with managing constipation in these patients. A prospective time and motion study also was undertaken, whereby staff recorded the time and resources required to perform each task. These data were then applied to the actual frequency recorded in the retrospective review to calculate the direct cost of managing constipation in those 70 admissions during that six-month period. There was no discernable pattern in oral laxative prescribing. The mean cost of managing constipation was 29.81 pounds (48.74 USD) per admission, with staff time accounting for 85% of the cost. The most time-consuming activity was staff discussion about bowel management, which occurred at least once daily for doctors and twice for nurses and involved up to eight members of staff at a time. The cost of managing constipation is skewed in that it costs 30 pounds (49 USD) or less in 71% of admissions but exceeded 100 pounds (163 USD) in 5%. In the latter group, earlier and/or more effective intervention for constipation could lead to clinical and economic benefits.