Role of water coordination at zinc binding site and its catalytic pathway of dizinc creatininase: insights from quantum cluster approach.

Creatininase is a key enzyme of creatinine-metabolizing pathway in mammals, and has a great potential for diagnostic application. It catalyzes the reversible conversion of creatinine to creatine. Here, we investigated its reaction mechanism with density functional theory in conjunction with the quantum cluster approach. Three reaction pathways in which several possible proton transfers assisted by either His178 or a water ligand to Zn1 (Wat2) or both were considered. DFT calculations reveal, depending on Wat2 coordination mode at Zn1, two competitive ring-opening pathways where His178 playing a central role as a proton shuttle or both His178 and Wat2 serving as a dual catalytic role as a base and an acid, respectively. Three elementary steps were proposed for the reaction: the first involves nucleophilic attack by a bridging hydroxide to the substrate and forms a gem-diolate intermediate, followed by a proton transfer from the gem-diolate to His178 (His178 protonation is a required step for efficient proton transfers). Finally, the second proton transfer from the protonated His178 or Wat2 to the amide of substrate leads to the ring opening. The first proton transfer is the rate-limiting step of the whole reaction, in consistent with previous experimental and computational studies. A detailed understanding of the reaction mechanism of the creatininase enzyme family will also be helpful for developing a biosensor for kidney function.

A novel assay for serum creatinine using a creatine kinase cycling reaction.

For assaying serum creatinine, the enzymatic method is regarded as accurate. However, a reliable measurement of low levels is problematic. We have developed a new method that utilizes an enzymatic cycling reaction involving creatine kinase (CK) in the presence of excess ATP and IDP and implicated the application to a serum creatinine assay by incorporating with creatininase. Here, we evaluated applying the CK cycling method to a serum creatinine assay. In this study, we focused on assessing whether an accurate measurement could be achieved, especially in the reference interval and the lower concentration range. The effective sensitivity of the assay using 30 U/mL CK was approximately 4-fold greater than that of a commercial reagent. Under these conditions, 0.19 mg/dL of creatinine was accurately detected. The correlation coefficient of the comparison study with an existing commercial reagent was 0.995. Moreover, the effect of the increased signal intensity on accuracy and precision was assured.

A new disposable microfluidic electrochemical paper-based device for the simultaneous determination of clinical biomarkers.

A new disposable microfluidic electrochemical paper-based device (ePAD) consisting of two spot sensors in the same working electrode for the simultaneous determination of uric acid and creatinine was developed. The spot 1 surface was modified with graphene quantum dots for direct uric acid oxidation and spot 2 surface modified with graphene quantum dots, creatininase and a ruthenium electrochemical mediator for creatinine oxidation. The ePAD was employed to construct an electrochemical sensor (based on square wave voltammetry analysis) for the simultaneous determination of uric acid and creatinine in the 0.010-3.0 µmol L-1 range. The device showed excellent analytical performance with a very low simultaneous detection limit of 8.4 nmol L-1 to uric acid and 3.7 nmol L-1 to creatinine and high selectivity. The ePAD was applied to the rapid and successful determination of those clinical biomarkers in human urine samples.

Effect of age on electrical nerve conduction in the somatosensory pathway and its correlation with somatometry and plasma concentrations of musculoskeletal enzymes in male rhesus monkeys (Macaca mulatta) held in captivity.

BACKGROUND: Somatosensory evoked potentials (SEPs) make it possible to obtain functional data on the activity of somatosensory pathway. OBJECTIVE: To evaluate the ontogeny of electrical nerve conduction in male rhesus monkeys using SEPs in correlation with the development of the musculoskeletal system based on somatometry and musculoskeletal enzymes. METHODS: Somatosensory evoked potentials of the medial and tibial nerves were performed, and somatometric measurements were obtained: total length, arm and forearm length, and thigh and calf length. Analysis of the musculoskeletal enzymes, lactic dehydrogenase, and creatininase was conducted using blood samples in 20 rhesus monkeys divided into 5 groups. RESULTS: Statistical analysis manifested a delay in the appearance of latencies as age increased. Also evident was a strong, direct relation between the lengths and the value of the latencies of the SEP, together with an inverse relation between the musculoskeletal enzymes. CONCLUSIONS: These findings contribute to standardizing this animal model in the neurophysiological sciences.

Consecutive hydrolysis of creatinine using creatininase and creatinase encapsulated in Saccharomyces cerevisiae spores.

OBJECTIVES: To achieve consecutive conversion from creatinine to urea and sarcosine using creatininase and creatinase encapsulated in spores of Saccharomyces cerevisiae. RESULTS: Creatininase encapsulated into the spore wall was produced and its specific activity was 3.4 ± 0.4 U/mg. By deletion of OSW2 gene, which causes a mild spore wall defect, the activity was increased to 10.9 ± 0.5 U/mg. Compared with soluble enzymes, spore-encapsulated creatininase was tolerant to environmental stresses; creatininase encapsulated in osw2∆ spores retained more than 90 % of the activity after treatment by SDS or proteinase K. Creatinase capsules could also be produced through spore encapsulation. The mixture of spores containing either creatininase or creatinase could mediate a two-step reaction to produce urea from creatinine; 5 mg spores produced 19 µmol urea in 10 min. Spores co-expressing creatininase and creatinase could also mediate the reactions more efficiently than the mixture of spores individually expressing each enzyme; the yield in 10 min was 38 µmol. CONCLUSIONS: Yeast spores can hold creatininase and creatinase simultaneously and catalyze the consecutive reactions.