Creatinase

Creatinase can catalyze the hydrolysis of creatine to produce urea and creatine. At present, the existence of creatinase is found in a variety of bacteria. Creatinine is the end product of phosphocreatine metabolism in the human body. It can be filtered by the kidneys and then excreted from the blood into the urine. The normal range of serum creatinine content is between 35 and 150 μM, but when there is a problem with kidney or muscle function, the content of creatinine will rise to 1000 μM. Therefore, the creatinine content of blood and urine can reflect the excretory function of the kidneys. Compared with chemical detection, enzymatic detection has more advantages. As a key enzyme for enzymatic detection of creatinine content, creatinase plays a very important role in the clinical detection of kidney function.

Figure 1. Protein structure of creatinase.

Sources

The main source of creatinase is microorganisms. When solving the problem of creatinine measurement method, it was found that some microorganisms can use creatinine as the main nitrogen and carbon source for growth. It was first discovered in 1937 that creatinine could be arthrobacter. Subsequently, it was found that strains of other genera could induce creatinase and accumulate in cells. These bacteria include Pseudomonas, Clostridium, Flavobacterium, Bacillus, Alcaligenes, and Paracoccus.

Structural research

The reported microbial creatinase consists of a single subunit or two identical subunits with a molecular weight in the range of 45 to 51 kD. For example, the molecular weight of creatinase of P. putida var. Naraensis is 94 kD, which is composed of two identical 47 kD subunits, each of which has a thiol group, which is closely related to the activity of creatinase Related; Creatinase in P. putida consists of two identical subunits, each with two domains, inferred Met (157) Ile (158) -Lys (159) -Ser (160), His (232), Glu (262) and Glu (358) may be important residues that catalyze the hydrolysis of creatine; the creatinase of Alcaligenes is a single subunit enzyme with a molecular weight of 51 kD. Molecular biology methods were used to clone and express the creatinase gene, and the recombinant enzymes were all single subunit enzymes with molecular weights ranging from 42 to 50 kD.

Effects of metal ions and chemical reagents on the activity of creatinase

The effect of metal ions and chemical reagents on the activity of creatinase has been reported that creatinase from different sources can be completely inhibited by 1 mM Hg²⁺, Cu²⁺, and Zn²⁺. Each subunit of the enzyme has a thiol group This group is closely related to the activity of creatinase. Although the reactions to metal ions vary, all CREs studied have no metal ion activator. In addition, the metal chelating agent has no effect on the activity of the enzyme, indicating that the enzyme is not a metalloenzyme.

Applications

Creatinase has been widely used in clinical determination of creatinine content. The production and excretion of creatinine in serum are relatively stable, so the determination of serum creatinine concentration can more accurately reflect glomerular function than serum urea concentration. Therefore, the accurate determination of serum creatinine is very important for clinical diagnosis, observation of therapeutic effect and prognosis. At present, most of the methods for measuring creatinine are emergency biochemical equipment (electrode method) and automatic biochemical equipment (enzymatic method or Jaffe rate method). These three methods have different measuring principles and are also affected by non-specific substances. In the early days, the commonly used creatinine detection methods in clinic were mainly chemical detection methods. This method was mainly based on the Jaffe reaction, that is, the creatinine methylene reacted with picric acid under alkaline conditions to produce a red complex. Its concentration can be determined by colorimetry. However, the chemical detection method is not specific and has poor sensitivity, which limits its application in clinical determination.

Reference

1. Kha and Wolfgang W. A highly sensitive amperometric creatinine sensor. Analytica Chimica Acta,1997,351: 151-158.