Hans Moellering

Determination of citrate with citrate lyase.

Abstract A method is described for the rapid determination of citrate in biological material which is based on the complete cleavage of the substance by purified preparations of citrate lyase in the presence of zinc ions. The assay of citrate lyase is also markedly improved by addition of zinc. The method was applied to a number of animal tissues and fruit juices. It is absolutely specific for citrate.

Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida.

The three-dimensional crystal structure of creatine amidinohydrolase (creatinase EC 3.5.3.3) from Pseudomonas putida, a dimeric enzyme with a molecular weight of 97,000, has been determined by multiple isomorphous replacement, averaging over the local dyad and restrained crystallographic refinement at 1.9 A with a crystallographic R-value of 17.7%. The asymmetric unit contains a dimer. The two chemically identical subunits consist of 403 residues each. A subunit is built up of two domains, a small N-terminal and a larger C-terminal domain. The small domain has a central seven-stranded beta pleated sheet with short helices on the outside. The large domain forms a six-stranded antiparallel beta half-barrel with helices on the outside. The two domains are connected by a segment that links two helices. The binding site of the competitive inhibitor carbamoyl sarcosine, a close analog of the substrate creatine, is located in the center of the large domain and partly covered by the small domain of the other subunit. The carbamoyl group is tightly co-ordinated to a water molecule, which presumably represents the nucleophile involved in hydrolysis of creatine. A catalytic mechanism is proposed on the basis of this structure.

Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.