Urease

Introduction

A nickel-dependant enzyme¹, urease is effectively produced by many organisms using urea as a waste product.² Animals, plants, bacteria, fungi and some algs alike synthesize urease, which is also a present as a soil enzyme.³ The breakdown of urea by urease is as follows:

(NH₂)2CO + H₂O+heat → CO_{2 + 2NH₃⁴}

More specifically, urease catalyzes the hydrolysis of urea to produce ammonia and carbamate; the carbamate produced is subsequently degraded by spontaneous hydrolysis to produce another ammonia and carbonic acid.⁵

The significance of urease is that the reaction it catalyses has very high endothermic properties, meaning that there will be a significant heat intake in the conduction of the process, making the enzyme ideal to conduct a cooling process. Aside from its relatively high endothermic function, the enzyme has been historically important in major scientific establishments in respective fields and has been under the scope of many researchers worldwide, making urease one of the most identified enzymes in the world.⁶

Background Information

Both, urea and ureases, represent important milestones in modern Biochemistry. Urea, isolated from human urine in 1773, was the first organic molecule synthetized in laboratory from inorganic compounds, by Wöhler in 1828. In 1926, James B. Sumner crystallized urease from jackbean (Canavalia ensiformis), the first protein crystal ever obtained, and his work was fundamental to prove that enzymes are indeed proteins.

Urease was purified at the year of 1828. Being the enzyme to breakdown the major nitrogenous waste product, urea, the enzyme reached the glory of being the first enzyme to purified. It was with the purification of urease that it was understood that enzymes were functional outside of the cell. Also, many groundbreaking discoveries were made after this discovery, making urease one of the most commonly known and investigated enzymes of today. Since 1828, urease has been scrutinized in studies and has been described in terms of structure, activity, property and functionality. Urease was also the first enzyme to be crystallized by James B. Sumner in the year of 1926. It was his achievement that got him the Nobel Prize in 1946 and the world the understanding that enzymes were structurally proteins.⁷ ⁸

Structure and Activity of Urease

All ureases have functionally and structurally similar properties.⁹ The structure complexes are not uniform, as there exist several chimeric structures, but in effect and complex activities the enzyme structures are have great similarity. The nickel-complex of all ureases are the main effector of the reaction complex. The ureases that will be utilized in our project originate from the species of two different kingdoms, from the bacterium specie Sporosarcina pasteurii and the fungus specie Endocarpon pusillum. The functional efficiency and characterization of these two urease species are assumed to be different. There is an abundant foundation for research on S. pasteruii but the Endocarpon pusillum is an urease that remains to be characterized.

S. pasteruii is a trimeric enzyme with three chimeric parts, tagged as UreA, UreB and UreC. Almost all bacterial ureases have a similar trimeric enzyme structure and have very close activity levels.[8] These three subunits contain the α, ß and γ active sites that work in coordination to structurally change in the presence of urea to breakdown the molecule.¹⁰ The effectivity of the S. pasteruii can be understood with the relative kinetic activity of the enzyme with its counterparts, the Soy-Bean Urease and Jack-Bean Urease. The kinetic degradation rate of SBU is 1/100 of S. pasteruii urease and this relative value is 1/14 for the JBU.[8] S. pasteruii urease has a significantly higher enzymatic kinetic activity in respect to other common ureases. Also, urea is abundant substrate meaning that this reaction can reach high activity levels through high substrate concentration. E. pusillum urease is a relatively newly discovered member of the urease family of enzymes. E. pusillum is a lichen-forming fungus species, making the expected structure of the urease similar to those of other fungal species.¹¹ The lack of characterization of the enzyme means that the initial characterization efforts may be being conducted in this study and that the primary data source may be found here.

Thermochemistry and Kinetic of Urease

The Gibbs Free Energy values for the enzyme is a relatively high value for a substrate of simple structure and common existence in nature.

Table 1¹²
	Total Protein Concentration	GFP Flurometric Measurement	GFP/TOTAL
Toehold	7,1	0,2705	0,038099
Toehold+trigger	3,5	9,031	2,5802

As it can be seen on the table, the G solvent values are relatively high to the breakdown of other simple molecules in terms of structure.

The kinetic speed of the breakdown process increases with temperature, reaching relatively high levels at higher temperatures.

Table 2¹²
T [K]	k_exptx10^-5s^-1	k_calcdx10^-5s^-1	k_Hcalcdx10^-5s^-1
333.15	0.0207	0.045	0.079
343.15	0.083	0.150	0.240
353.15	0.385	0.282	0.724
363.15	1.20	0.525	2.089
373.25	4.15	1.59	6.30

The increasing kinetic activity with an increase in temperature also assist the needed alacrity in cooling as the temperature increases.

Citations