Urease crystallization

The isolation and crystallization of urease by James Sumner in 1926 provided a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes are proteins. In the absence of other examples, this idea remained controversial for some time. Only in the 1930s was Sumner s conclusion widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. During this period,... 

High resolution structures of urease from B. pasteurii complexed with /3-mercaptoethanol (13) (BME Fig. IB) and acetohydroxamic acid (14) (AHA Fig. 1C) indicate the presence of bridging/chelating coordination to the Ni ions, highlighting the importance of both metal ions in the reactivity of the enzyme. The structures of B. pasteurii urease crystallized in the presence of phosphate... 

Laufberger had tried to obtain the protein from horse liver, but it did not crystallize, and as he described to me when I met him in Prague some years ago, in those days everyone wanted to have protein crystals as a criteria of purity. Although James Sumner had crystallized jack bean urease in 1926, his preparations were somewhat impure, and it was only in the mid-1930s, when John Northrop and Moses Kubnitz showed that there is a direct correlation between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin that the protein nature of enzymes was generally accepted. 

Historically the earliest Ni-containing enzyme to be described was urease from jack bean meal, which was crystallized by James Sumner in 19261. However, analytical techniques did not allow urease to be recognized as a Ni-containing enzyme until 50 years later. Urease catalyses the hydrolysis of urea to ammonia and carbamate, which spontaneously hydrolyses to give carbonic acid and a second molecule of ammonia. 

James Sumner received the Nobel Prize for Chemistry in 1946 for the crystallization of proteins. Richard Willstatter, the 1915 Chemistry prizewinner, had proposed that proteins were not enzymes, and that the protein in urease was simply a scaffold for the veritable catalyst. Since urease is inactive without Ni, he was not so far wrong ... 

Hevesy first used a radioisotope as a tracer. Warburg. Importance of iron pigments in oxidation. Keilin rediscovered cytochromes. Sumner crystallized urease. The Eggletons and Fiske and SubbaRow isolated phospho-creatine. 

The crystal structure of urease form Klebsiella aerogenes has recently been determined (47). The two nickel(II) ions in the active site are... 

In 1926, James Summer crystallized the enzyme urease (Chapter 16) and crystallization of other enzymes soon followed.380 In 1934, J. B. 

Urease, which was first isolated from the jack bean has a special place in biochemical history as the first enzyme to be crystallized. This was accomplished by J. B. Sumner in 1926, and although Sumner eventually... 

Nickel has long been suspected to be an essential trace element for living organisms, but the identification of its functions in molecular terms is relatively recent. The first nickel protein to be identified was urease (urea ammonia hydrolase) (i). This was demonstrated 49 years after the original isolation and crystallization of the enzyme by Sumner (2). This enzyme is of widespread occurrence, and the specific requirement for nickel explains many of the effects of nickel deficiency in plants (3, 4). 

The title retains the trivial name for enzymes with the systematic name of urea amidohydrolase and the Enzyme Commission code number of EC 3.5.1.5. Ureases are hydrolases acting on C-N bonds (nonpeptide) in linear amides and thus belong to a group that includes glutaminase, form-amidase, and formyltetrahydrofolate deformylase. The title is plural to emphasize that urease activity may be exhibited by several protein species. Urease, singular, has come to mean by common usage, that particular enzymic protein first crystallized by Sumner from jack bean... 

Urease activity in soils has been found to reflect the bacterial count and content of organic matter. The urease isolated from an Australian forest soil (87) was crystallized and found to have a specific activity of 75 Sumner units (S.U.) per mg. The molecular weight species were estimated (sedimentation velocity) to be 42, 131, and 217 X 103. That urease activity persists in soils is shown by the finding that enzymic activities, including urease, could be demonstrated in soil samples over 8000 years old (88). 

Much uncertainty reigned over the nature of proteins, the best known of which were hemoglobin, the digestive enzymes, and later, insulin. Properties of individual amino acids and the peptide bond were studied early in this century, but it was not until urease was crystallized by Sumner1 in 1926, followed by the isolation of other pure enzymes, that it was finally accepted in the 1930s that enzymes were proteins and that their catalytic properties were not the function of some adsorbed low molecular weight entity. Somewhat later, towards the end of the 1930s, coenzymes were isolated and their roles established. 

James B. Cornell Univ., 1st enzyme crystallized urease from... 

Urease (urea amidohydrolase) is an enzyme first identified over a hundred years ago in bacterial extracts [22], The presence of urease is a virulence factor for some pathogenic bacteria [23,24], It is now known to occur also in plants, fungi, and invertebrates (see [24,25] for reviews). Urease from jack bean was the first enzyme to be crystallized, in 1926. Almost 50 years later its metal content was reexamined and it was found to contain two atoms of nickel per subunit [26]. Finally in 1995 the crystal structure of the enzyme from the enteric bacterium Klebsiella aerogenes was determined [27], Amino-acid sequence comparisons predict that the structures of the plant and bacterial enzymes are similar, although with different subunit arrangements. 

A clearer picture emerged when the crystal structure of K. pneumoniae urease was determined [27], The nickel atoms in the center, Ni-1 and Ni-2, are 3.5 A apart. They are bridged by a carbamyl group, formed from C02 and a lysine residue, explaining the requirement for hydrogen carbonate in reconstitution. The other ligands are two histidines for Ni-1 and an aspartate and two histidines for Ni-2. 

From the crystal structure of urease, Jabri et al. [27] proposed that urea binds through its carbonyl oxygen, whereas the -NH2 hydrons are hydrogen-bonded to residues in the protein (Figure 1). The structure of the site is such that water molecules in the active site do not coordinate optimally to the nickel ions in the substrate-free form. As a result, the binding of urea is favored [40], A loop of polypeptide forms a flap that covers the active site once urea is bound. This flap includes cysteine 319, which had been believed to be catalytically important [41] and is one of the residues proposed to hydrogen-bond to the urea nitrogens. Mutation of this cysteine to alanine leads to decrease, but not necessarily loss, of activity. 

Continuous studies were performed in specially prepared microreactors molded from PDMS, designated PDMS (Sylgard 184 silicone elastomer Dow Corning) poured onto silicon wafer molds. The microreactor molds were prepared using 4-in. silicon wafers of Type P, crystal orientation of , resistivity of 1 to 2 Q, and thickness of 457-575 pm from Silicon Quest (Santa Clara, C A). After preparation, mixtures of urease enzyme and PDMS (designated PDMS-E) were poured onto the microreactor mold and allowed to cure at ambient conditions. 

The proteins from the jack bean (Canavala ensiformis) were first studied over 60 years ago by Jones and Johns(6). Several years later, Sumner(7), while studying urease (also from the jack bean), isolated three other proteins, two of which could be crystallized, concanavalin A and B. It should be noted that this report of crystalline Con A by Sumner appeared about seven years before he reported the first crystallization of an enzyme, urease(8). 

This reaction is catalyzed by a highly specific enzyme, urease. Urease is present in a number of bacteria and plants. The most common source of the enzyme is jack bean or soybean. Urease was the first enzyme that was crystallized. Sumner, in 1926, proved unequivocally that enzymes are protein molecules. 

The chemical nature of enzyme was controversial for a long time, until Buchner succeeded in isolating an enzyme system (zymase) from yeast in a cell-free extract in 1897.2) Urease was then crystallized by Sumner in 1926,3) followed by crystallization of several proteolytic enzymes by Northlop and his colleagues. At present the chemical nature of enzyme is defined as a protein with catalytic activity based on the specific activaiton of its substrate. However, this definition has been somewhat open to debate since a catalytic RNA, ribozyme, was discoved in 1982. 

Prior to the crystallization of jack bean urease it was assumed by the biochemical community that enzymes had no ordered structure. In 1965 the first crystallographic evidence for the mechanism by which enzymes work when Phillips and his group solved the lysozyme structure [6], Details of the structure indicated how the enzyme could bind the oligosaccharides present in its target, bacterial cell wall peptidoglycans, and could respond to the binding event by changing its structure. 

Jabri E et al (1995) The crystal structure of urease from Klebsiella aerogenes. Science 268 998-1004 PDBID 2KAU... 

Metalloenzymes pose a particular problem to both experimentalists and modelers. Crystal structures of metalloenzymes typically reveal only one state of the active site and the state obtained frequently depends on the crystallization conditions. In some cases, states probably not relevant to any aspect of the mechanism have been obtained, and in many cases it may not be possible to obtain states of interest, simply because they are too reactive. This is where molecular modeling can make a unique contribution and a recent study of urease provides a good example of what can be achieved119 1. A molecular mechanics study of urease as crystallized revealed that a water molecule was probably missing from the refined crystal structure. A conformational search of the active site geometry with the natural substrate, urea, bound led to the determination of a consensus binding model[I91]. Clearly, the urea complex cannot be crystallized because of the rate at which the urea is broken down to ammonia and, therefore, modeling approaches such as this represent a real contribution to the study of metalloenzymes. 

Many enzymes were purified from a large number of sources, but it was J. B. Sumner who was the first to crystallize one. The enzyme was urease from jack beans. For his travail, which took over 6 years (1924-1930), he was awarded the 1946 Nobel prize. The work demonstrated once and for all that enzymes are distinct chemical entities. 

Historically, the first chemical synthesis of urea by Wohler, from ammonium cyanate in 1828, was a milestone that established a bridge between inorganic and organic chemistry. Urease was the first enzyme ever to be crystallized (6), and it was the first protein shown to contain nickel ions in the active site (7). The first X-ray crystal structure of urease became known in 1995 (8). Significant progress was made since then toward an understanding of its catalytic mechanism, as well as toward the structural and functional emulation of its active site by synthetic model complexes (5, 9). 

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