Hans Mollering

Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2·0Åresolution

N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetramer of 116,000 M(r) in the asymmetric unit. The structure determination proceeded by multiple isomorphous replacement, followed by solvent-flattening and density averaging about the local diads within the tetramer. In the final refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.7 degrees for bond angles. The asymmetric unit consists of 7853 protein atoms, 431 water molecules and four sulfate ions bound into the putative active site clefts in each subunit. One subunit contains a central six-stranded parallel beta-pleated sheet packed by helices on both sides. On one side, two helices face the solvent, while two of the helices on the other side are buried in the tight intersubunit contacts. The catalytic center of the enzyme, tentatively identified by inhibitor binding, is located at the interface between two subunits and involves residues from both. It is suggested that the nucleophilic group involved in hydrolysis of the substrate is the thiol group of Cys117 and a nucleophilic addition-elimination mechanism is proposed.

Fully enzymatic colorimetric assay of serum and urine creatinine which obviates the need for sample blank measurements

Abstract A fully enzymatic method for the colorimetric determination of serum and urine creatinine is described which does not require sample blank measurements. It is based on the formation of hydrogen peroxide from creatinine in a reaction sequence catalyzed by creatinine iminohydrolase, ATP-dependent 1-methylhydantoinase, N-carbamoylsarcosine amidohydrolase and sarcosine oxidase. The hydrogen peroxide is quantitated with high sensitivity at 546 nm by a chromogenic system consisting of peroxidase, 2′-sulpho-2-methyl-benzthiazolinone hydrazone and 2,4,6-tribromo-3-hydroxy-benzoic acid. Only 20 μL of sample are needed for the assay, the total reaction being complete within 10 min at 25°. Within-run precision gave a CV of 3.1 and 1.6 % at serum creatinine concentrations of 79 and 160 μmol/L, respectively, and the standard curve is linear up to at least 1760 μmol/L. The assay yields results which agree well with those found by both an enzymatic UV-method and an alternate enzymatic colorimetric procedure nec...

Analytical Differentiation of Purine and Pyrimidine Nucleotides Determination of ADP, ATP, and Sum of GTP + ITP in Biological Material

Publisher Summary This chapter presents the determination of adenosine-5′-diphosphate (ADP), adenosine-5′-triphosphate (ATP), and the sum of guanosine-5′-triphosphate (GTP) and ionosine-5′-triphosphate (ITP) in biological material. The specific enzymatic determinations of ATP, for example by using luciferase, have been known for some time; however, in the past, ATP has been determined mainly by two non-specific methods: (1) with hexokinase (HK) and glucose-6-phosphate dehydrogenase and (2) with phosphoglycerate kinase (PGK) and glyceraldehyde phosphate dehydrogenase (GAPDH). HK and PGK act on the other nucleoside triphosphates as well as on ATP. Only myokinase (MK) is specific for ADP and ATP. When nucleoside triphosphates are converted non-specifically into the diphosphates by any kinase, only ADP, out of all the diphosphates, can be specifically converted by myokinase into ATP and AMP. This chapter describes a method in which the creatine kinase (CK) reaction is used for the specific determination of the ADP originally present in the sample. ATP inhibits the F-6-PK reaction, but this inhibition can be eliminated by the addition of A-5-MP.