An initial study to develop methodologies to examine catalytic mechanisms in enzymatic reactions from the urease superfamily

In an effort to standardize nomenclature, a committee of the International Union of Biochemistry (IUB) has published and maintained a conventional of classification scheme that categorizes enzymes into six major groups according to the general class of organic chemical reactions they catalyze. The IUB classification scheme assigns a unique code number (E.C. x.x.x.x.) to each enzyme identifying the major class, the subclass, and its specific reaction.;One particular subclass in interest, aminohydrolyase (E.C. 3.5.x.x.), includes a number of enzymes catalyzing the hydrolysis of a given amino compounds. The IUB system classifies these enzymes into a group of aminohydrolyases, but there is a closer relationship between two of these enzymes that the IUB system cannot indicate and distinguish them from other aminohydrolyases.;Urease (E.C. 3.5.1.5.) and Adenosine Deaminase (E.C. 3.5.4.4.) are related to each other beyond the level of merely catalyzing the same type of reaction. They have highly resembled active site architectures, and it is generally believed that they exhibit the same pattern of reaction mechanism.;In the analysis of purine utilization and metabolism in Methanobacterium thermoautotrophicum strain DeltaH (previous work by Dr. Christine Clougherty), a methanogenic member of the Archaebateria kingdom, an interesting phenomenon was observed where the cell growth was inhibited by extracellular presence of adenosine. The deaminated product, inosine, was subsequently detected in the media. There was no rational explanation until the idea of urease superfamily was introduced.;We speculate the enzyme that guarding the front door of nitrogenous acquisition pathway in this organism is an unique aminohydrolase, possibly a derivative urease and competitively inhibited by adenosine. Granted, native ureases from bacterium or eucaryotic sources are unlikely to see adenosine as inhibitor, but the same argument may be validated when it comes down to archeabacteria.;In this project we investigated two major categories characterized as the key elements conserved among members of the urease superfamily. The investigations were conducted on native urease from jack bean plant and adenosine deaminase from calf intestinal mucosa. We interpreted the results into guidelines to analyze the similarity as well as the diversity between the catalytic mechanisms of urease and adenosine deaminase, and we were aiming on applying the same guideline to examine that special aminohydrolase in Mb. t. strain DeltaH.;However, we have not been able to reproduce the "adenosine phenomenon" described in Dr. Clougherty's report. At this point we had two brands of Mb. t. strain DeltaH sources and we found them reacted quite differently upon the extracellular presence of adenosine in the recent growth studies. Before a further detailed investigation can be completed, we can only assume that a mutation or a sub-strain of Mb. t. DeltaH was involved, and the properties of our target aminohydrolase in his organism had been altered.