Penicillin, enzymatic reactions

J. L. Strominger, Enzymatic Reactions in Bacterial Cell Wall Synthesis Sensitive to Penicillins, Cephalosporins, and Other Antibacterial Agents , Antibiotics 1967, 1, 705-713. 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

Another interesting example of resolution through formation of diastereo-mers is the isolation of four stereoisomers of 3-amino-2-methyl-3-trifluoro-methyl butanoic acid [55]. In this process, the chemical-enzymatic method by the combination of chemical and enzymatic reaction is a very convenient. At first, -phenylacetyl derivatives 61a and 61b were prepared in excellent isolated yields via the Schotten-Baumann procedure. After these materials were hydrolysed with penicillin acylase (EC 3.5.1.11) from Escherichia coli until attainment of 50% conversion, enzymatically unconverted -phenylacetyl derivatives 62 a and 62 b (organic layer) and amino acids 63 b and 63 d (aqueous layer) were separated. Acidic hydrolysis of unconverted materials produced other stereoisomers 63 a and 63 c in high optical pure form. 

It has been proposed that penicillin has a conformation which is similar to the transition-state conformation taken up by D-Ala-D-Ala—the portion of the amino acid chain involved in the cross-linking reaction (Fig. 10.61). Since this is the reaction centre for the transpeptidase enzyme, it is quite an attractive theory to postulate that the enzyme mistakes the penicillin molecule for the D-Ala-D-Ala moiety and accepts the penicillin into its active site. Once penicillin is in the active site, the normal enzymatic reaction would be carried out on the penicillin. 

The inhibition of bacterial cell wall synthesis by penicillin is a classic example of a medically significant inhibition of an enzymatic reaction. Which of the following statements about the inhibition of glycopeptide transpeptidase by penicillin is true ... 

Because of the usual lag time between sample collection and analysis at the laboratory, sample storage is an important step. The potential effects of physicochemical factors such as oxidation, proteolysis, and precipitation and biological factors that include microbiological and enzymatic reactions need to be considered when storing samples. For example, the production of the enzyme penicillinase, which is capable of reducing the concentration of penicillin in kidney tissue stored at 4°C, has been reported in some studies. Preservation can be achieved through the addition of enzyme inhibitors (e.g., piperonylbutoxide inhibits cytochrome P450). 

Industrial applications represent more than 80% of the global market of enzymes. A distinction should be made between those cases in which the enzymatic conversion of the raw material into the product is the key operation and those in which the enzyme is used as an additive to modify certain functional property of the product. In the first case the enzymatic reaction is carried out in a controlled environment at optimized conditions with respect to the catalytic potential of the enzyme, while in the second case conditions for enzyme action are not specified to optimize its activity and sometimes not even controlled. Examples of the first case are the production of high-fructose syrups with immobilized glucose isomerase and the production of 6-aminopenicillanic acid from penicillin G with immobilized penicillin acylase examples of the second case are the use of fungal proteases in dough making and the use of pancreatin in leather bating. Most conventional uses of enzymes refer to... 

Fig. 27. Activity loss a/a of Acylase enzym resign with the reaction time t of the enzymatic deaccylation of Penicillin G to 6-Aminopenicillanic acid (reactor design see Fig. 28)...

Drugs can also Interfere with laboratory results by negating certain nonspecific oxidation and reduction reactions essential for the chemical assay. Penicillin, streptomycin and ascorbic acid are known to react with cupric Ion thus, false positive results for glucose may occur If a copper reduction method Is used. If the specific enzymatic glucose-oxidase method Is employed, ascorbic acid can cause a false negative result by preventing the oxidation of a specific chromogen In the reaction. 

Enzymes are the catalyst per excellence for reactions in water, which is their natural habitat. Moreover, the use of enzymes often circumvents the need for functional group protection and deprotection steps. For example, enzymatic hydrolysis of penicillin G to 6-APA (Fig. 2.30) proceeds in one step at ambient temperature while chemical deacylation requires three steps, a temperature of - 40 C and various stoichiometric reagents, leading to a high E factor. 

DKPs are simple and easy to obtain and are quite common by-products of synthetic, spontaneous, and biological formation pathways. DKP formation has been well documented as side reactions of solid-phase and solution-phase peptide synthesis. In addition, DKPs have been shown to be decomposition products of various peptides, proteins, and other commercial pharmaceuticals. Cyclic dipeptides were found to be present in solutions of human growth hormone, bradykinin, histerlin, and solutions of agents within the classes of penicillins and cephalosporins. " DKPs are also enzymatically synthesized in several protists and in members of the plant kingdom. Hydrolysates of proteins and polypeptides often contain these compounds and they are commonly isolated from yeasts, lichens, and fungi. ... 

Drug/Lab test interactions False-positive urine glucose reactions may occur with penicillin therapy if Clinitest, Benedict s Solution, or Fehling s Solution are used. It is recommended that enzymatic glucose oxidase tests (such as Clinistix or Tes-Tape) be used. Positive Coombs tests have occurred. High urine concentrations of some penicillins may produce false-positive protein reactions (pseudoproteinuria) with the P.870... 

In addition, at very low water contents, ampicillin accumulation curves do not exhibit a clear-cut maximum, inherent in the enzymatic acyl transfer reactions in aqueous medium (including quite concentrated heterogeneous aqueous solution-precipitate systems), because of the secondary hydrolysis of the target product by penicillin acylase (Figure 12.6) [84]. 

Although DTT is not necessary in the enzymatic conversion of penicillin G [14], this compound is known to stimulate the expandase activity of frozen crude extracts of S. clavuligerus and C. acremonium on penicillin N [40], However, neither this reagent nor P-mercaptoethanol was able to stimulate the subsequent reaction with penicillin G when added during preincubation with FeS04 and ascorbate [43],... 

---