RESUMEN
Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with Ki values ranging from 0.29 to 0.6 mM and KM values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger KM values between 83 and 260 mM under the same conditions. The kcat values ranged from 0.5 to 2.5s-1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s-1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule.
RESUMEN
PURPOSE: Examine and identify the breadth of definitions and measures of objective and subjective spinal stiffness in the literature, with a focus on clinical implications. METHODS: A scoping review was conducted to determine what is known about definitions and measures of the specific term of spinal stiffness. Following the framework by Arksey and O'Malley, eligible peer-reviewed studies identified using PubMed, Ebsco health, and Scopus were included if they reported definitions or measures of spinal stiffness. Using a data abstraction form, the studies were classified into four themes: biomechanical, surgical, pathophysiological, and segmental spinal assessment. To identify similarities and differences between studies, sixteen categories were generated. RESULTS: In total, 2426 records were identified, and 410 met the eligibility criteria. There were 350 measures (132 subjective; 218 objective measures) and 93 indicators of spinal stiffness. The majority of studies (n = 69%) did not define stiffness. CONCLUSION: This review highlights the breadth of objective and subjective measures that are both clinically and methodologically diverse. There is no consensus regarding a standardised definition of stiffness in the reviewed literature.
There is a need for a consensus definition of stiffness, as we did not find a consensus definition of stiffness in the reviewed literature.Definitions of stiffness should be included alongside self-reported or patient-reported outcome measures.There is a need to establish the relationship between subjective and objective measures in future studies.Clinicians should be aware that stiffness is a symptom that may indicate the presence of underlying pathophysiology.
