Specificity and Kinetics of Haloalkane Dehalogenase

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the “natural” substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1,2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state kcat with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Clα) from 1,2-dichloroethane interacts with Trp125 and the Clβ interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in kcat could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The kcat for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Clα or Brα) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Real-time feedback to reduce low-back load in lifting and lowering

Low-back pain (LBP) is a common health problem. Literature indicates an exposure-response relation between work-related lifting and LBP. Therefore, this study investigated effects of three kinds of real-time feedback on low-back load, quantified as lumbar moments, during lifting. We recruited 97 healthy male and female participants without a recent history of LBP and without prior biomechanical knowledge on lifting. Participants were assigned to groups based on the time of enrollment, filling the four groups in the following order: moment feedback, trunk inclination angle feedback, lumbar flexion feedback, and a control group not receiving feedback. Feedback was given by a sound when a threshold level of the input variable was exceeded. Participants were unaware of the input variable for the feedback, but were instructed to try to avoid the audio feedback by changing their lifting strategy. The groups with feedback were able to reduce the audio feedback and thus changed the input variable towards a more desired level. Lumbar moments significantly decreased over trials in the inclination and moment feedback groups, remained similar in the lumbar flexion group and increased in the control group. Between group comparisons revealed that low-back load was significantly lower in the moment and inclination groups compared to the control group. Additionally, moments were lower in the inclination group than in the lumbar flexion group. Real-time feedback on moments or trunk inclination is a promising tool to reduce low-back load during lifting and lowering

Predicting the influence of hip and lumbar flexibility on lifting motions using optimal control

Computational models of the human body coupled with optimization can be used to predict the influence of variables that cannot be experimentally manipulated. Here, we present a study that predicts the motion of the human body while lifting a box, as a function of flexibility of the hip and lumbar joints in the sagittal plane. We modeled the human body in the sagittal plane with joints actuated by pairs of agonist-antagonist muscle torque generators, and a passive hamstring muscle. The characteristics of a stiff, average and flexible person were represented by co-varying the lumbar range-of-motion, lumbar passive extensor-torque and the hamstring passive muscle-force. We used optimal control to solve for motions that simulated lifting a 10 kg box from a 0.3 m height. The solution minimized the total sum of the normalized squared active and passive muscle torques and the normalized passive hamstring muscle forces, over the duration of the motion. The predicted motion of the average lifter agreed well with experimental data in the literature. The change in model flexibility affected the predicted joint angles, with the stiffer models flexing more at the hip and knee, and less at the lumbar joint, to complete the lift. Stiffer models produced similar passive lumbar torque and higher hamstring muscle force components than the more flexible models. The variation between the motion characteristics of the models suggest that flexibility may play an important role in determining lifting technique

Effects of a passive back exoskeleton on the mechanical loading of the low-back during symmetric lifting

Low-back pain is the number one cause of disability in the world, with mechanical loading as one of the major risk factors. Exoskeletons have been introduced in the workplace to reduce low back loading. During static forward bending, exoskeletons have been shown to reduce back muscle activity by 10% to 40%. However, effects during dynamic lifting are not well documented. Relative support of the exoskeleton might be smaller in lifting compared to static bending due to higher peak loads. In addition, exoskeletons might also result in changes in lifting behavior, which in turn could affect low back loading. The present study investigated the effect of a passive exoskeleton on peak compression forces, moments, muscle activity and kinematics during symmetric lifting. Two types (LOW and HIGH) of the device, which generate peak support moments at large and moderate flexion angles, respectively, were tested during lifts from knee and ankle height from a near and far horizontal position, with a load of 10 kg. Both types of the trunk exoskeleton tested here reduced the peak L5S1 compression force by around 5-10% for lifts from the FAR position from both KNEE and ANKLE height. Subjects did adjust their lifting style when wearing the device with a 17% reduced peak trunk angular velocity and 5 degrees increased lumbar flexion, especially during ANKLE height lifts. In conclusion, the exoskeleton had a minor and varying effect on the peak L5S1 compression force with only significant differences in the FAR lifts