Urease substrate binding

Type (820) dinickel complexes offer the opportunity of substrate binding within the bimetallic pocket, and highly preorganized complexes of this type have also been employed as model systems for the urease metalloenzyme (see Section 6.3.4.12.7). The Ni—Ni separation in type (820) complexes can be... 

Hydration and/or dehydration reactions are frequently catalyzed by metallopro-teins. Examples are proteins containing nickel (urease), zinc (e.g., peptidases), molybdenum (the hydratase partial reaction of formate oxidoreductase), tungsten (acetylene hydratase). An obvious difference between Ni, Zn, on the one hand, and Fe, Mo, W, on the other, is that the first are directly coordinated to the protein whereas the latter are also part of a cofactor. With reference to the Fe/S cluster in aconitase it has been suggested that cofactor coordination may provide an added flexibility to the active site, in particular to the substrate binding domain [15],... 

Many chemical reactions in living systems are catalyzed by enzymes. An enzyme is a large protein molecule (typically of molar mass 20,000 g moP or more) with a structure capable of carrying out a specific reaction or series of reactions. One or more reactant molecules (called substrates) bind to an enzyme at its active sites. These are regions on the surface of the enzyme where the local structures and chemical properties will selectively bind a specific substrate so particular chemical transformations of it can be carried out (Fig. 18.18). Many enzymes are quite specific in their active sites. The enzyme urease catalyzes the hydrolysis of urea, (NHzlzCO,... 

Urease has a molecular weight of590 000 30 000 and consists of six identical subunits. Each subunit contains two Ni ions of different valency which are involved in substrate binding and conversion. The isoelectric point of the protein is at pH 5 and the temperature optimum of the catalysis at 60°C. The kinetic constants for urea hydrolysis have been determined to be k+2 = 5870 s"1 and Km = 2.9 mmol/1. Other amides, such as formamide and semicarbazide, react much more slowly than urea. The pH optimum of urease depends on the nature of the buffer used and, with the exception of acetate buffer, equals the pJTs value of the buffer. The active center of urease contains an SH-group that is essential for the stability of the enzyme. Complexing agents, such as EDTA and reductants, are required for stabilization. 

Dixon et al. (35) have proposed a mechanism for urease catalysis (Fig. 3) based on studies of the reactions with the poor substrates formamide, acetamide, and iV-methylurea. They suggest that the two nickel ions are both in the active site, one binding urea and the other a hydroxide ion which acts as an efficient nucleophile. This implies that the nickel ions are within 0.6 nm (1 nm = 10 A) of each other so far it... 

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. 

The structure of the complex of urease with urea in the active site is unknown, because the enzyme-substrate intermediate is very short-lived and has not been trapped. Nevertheless, a number of inhibitors of urease that bridge between the nickel atoms are known. Acetohydroxamate is the most studied and binds slowly but with high affinity (K = 4 vaM [25]). Phosphoroamide is also a slowly binding inhibitor. 2-Thioethanol causes the appearance of sulfur-to-nickel... 

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. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

In addition, urease is inhibited by a variety of agents including fluoride and disulfldes, as observed recently in the acetohydroxamate-inhibited C319A variants of K. aero-genes urease. It is proposed that this mode of inhibition involves coordination of the inhibitor to only one nickel center, while another involves the inhibitor bridging the dinickel center. Urease is slowly inhibited by fluoride in both the presence and absence of substrate, and fluoride binding rates are directly proportional to inhibitor concentration. Fluoride inhibition is pH-dependent due to a protonation... 

Nickel is required for the synthesis of active urease in plant and other cells. The enzyme catalyzes the hydrolysis of urea to carbon dioxide and ammonia, via the intermediate formation of carbamate ion (equation 46). The molecular weight has been redetermined recently as 590 000 30 000, with six subunits. Each subunit has two nickel centres and binds one mole of substrate. The activity of the enzyme is directly proportional to the nickel content, suggesting an essential role for nickel in the enzyme. Several approaches, including EXAFS measurements, suggest that histidine residues provide some ligands to nickel, and that the geometry is distorted octahedral. There appears to be a role for a unique cysteine residue in each subunit out of the 15 groups present. Covalent modification of this residue blocks the activity of the enzyme. 

Binding of the substrate urea to a nickel ion in urease is an integral part of the mechanism in the hydrolysis reaction (Nielsen 1984). Both ruminants and monogastric animals require urease for the decomposition of urea into ammonia, which is needed for the microbial synthesis of ammonia that, in turn, is necessary for amino acid and protein synthesis. This process also takes place in the appendix of monogastric animals and some species of ruminants (roe deer). 

The link between enzymes and substrates is so strong that enzymes often are named after the substrate involved, simply by adding ase to the name of the substrate. Eor example, lactase is the enzyme that catalyzes the breakdown of lactose, while urease catalyzes the chemical breakdown of urea. Enzymes bind their reactants or substrates at special folds and clefts, named active sites, in the structure of the substrate. Because numerous interactions are required in then-work of catalysis, enzymes must have many active sites, and therefore can have molecular weights as high as one million. 

In noncompetitive inhibition, the inhibitor is presumed not to bind to an active site on the enzyme, but rather to bind at some other site. This complex formation may involve some change in the conformation of the enzyme, which makes it impossible for the substrate to bind at the active site. The inhibition of urease by Ag+, Pb2+, or Hg2+ is believed to be the result of these metal ions binding to the sulfhydryl (—SH) groups on the enzyme. For this type of action, we can write the equilibria... 

---