Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change

A clinical prediction rule for classifying patients with low back pain who demonstrate short-term improvement with mechanical lumbar traction

The objective of the study was to develop a clinical prediction rule for identifying patients with low back pain, who improved with mechanical lumbar traction. A prospective, cohort study was conducted in a physiotherapy clinic at a local hospital. Patients with low back pain, referred to physiotherapy were included in the study. The intervention was a standardized mechanical lumbar traction program, which comprised three sessions provided within 9 days. Patient demographic information, standard physical examination, numeric pain scale, fear-avoidance beliefs questionnaire and Oswestry low back pain disability index (pre- and post-intervention) were recorded. A total of 129 patients participated in the study and 25 had positive response to the mechanical lumbar traction. A clinical prediction rule with four variables (non-involvement of manual work, low level fear-avoidance beliefs, no neurological deficit and age above 30 years) was identified. The presence of all four variables (positive likelihood ratio = 9.36) increased the probability of response rate with mechanical lumbar traction from 19.4 to 69.2%. It appears that patients with low back pain who were likely to respond to mechanical lumbar traction may be identified