New Enzymes

Enzymes, nature s catalysts, are very attractive to chemical companies Enzymes are very efficient catalysts and as natural products they tend to be easier on the environment than human-made catalysts. However, one disadvantage of most enzymes is that they can function only near room temperature and at pH values near 7. Because of these limitations scientists are now looking at the enzymes that occur in organisms that exist in extreme conditions. 

A micrograph of the extremophile Archaeoglobus fulgidis, an organism that lives in the hot sediments near submarine hydrothermal vents. 

The DNA fragments are expressed (proteins are made from the genes contained in the fragments) most commonly using E. coli. Interestingly, even though E. coli must be cultured under the mild conditions necessary for them to survive, the extremozymes formed seem to have their characteristic catalytic activities, indicating that they have the same structures as when they are formed in their native extreme conditions. 

The mixtures of extremozymes produced by the expression process are then tested to see if they have catalytic activity for the industrial processes of interest. If a given mixture shows catalytic activity, it is then usually subjected to random DNA mutagenesis or molecule breeding to see whether random evolution of the enzymes will lead to improved activities. 

In the upper atmosphere the presence of nitric oxide has the opposite effect—the depletion of ozone. The series of reactions involved is 

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It is likely that any new enzymes isolated by screeners will be quickly and routinely cloned by genetic engineers, and be sequenced and expressed as almost pure proteins. Protein chemists can then evaluate the properties of the new enzyme and determine its three-dimensional stmcture. This vast amount of information allows the protein engineers and their computers to design the enzymes of the future. 

A system based partly on historical names, partly on the substrate, and partly on the type of reaction catalyzed is far from satisfactory. In 1956, the International Union of Biochemistry set up a Commission on Enzymes to consider the classification and nomenclature of enzymes. The Commission presented a report in 1961 whose recommendations for naming and classifying enzymes were subsequently adopted (12). Enzymes are classified on the basis of the reactions they catalyze. Despite its apparent complexities, the system is precise and very descriptive, accommodating existing enzymes and serving as a systematic basis for the naming of new enzymes. AH enzymes are placed in one of the six principal classes. 

Alpha/beta barrels provide examples of evolution of new enzyme activities... 

These results are compatible with an evolutionary history in which the new enzyme activity of mandelate racemase has evolved from a preexisting enzyme that catalyzes the basic chemical reaction of proton abstraction and formation of an intermediate. Subsequent mutations have modified the... 

W. Hummel, in New Enzymes for Organic Synthesis. Screening, Supply and Engineering (T. Scheper, ed.) [Advances in Biochemical Engineering. Biotechnology, Vol. 58], p. 145. Springer-Verlag, Berlin, 1997. 

When the activity and other properties of the several times recrystallized new enzyme protein are compared with those of the uncrystallized precipitate obtained in the first stages of the process, it Is found that even in the first stages, the enzyme is present in sufficiently pure form for most purposes. 

Many of these have been demonstrated with a range of antibiotics and antibiotic precursors, although relatively few have been applied commercially. We have included a list of published examples in the form of an Appendix at the end of this chapter. We do not expect you to remember the details of this Appendix. It has been included as an potential for illustration of the potential to use enzymes to modify organic molecules like antibiotics. Usin9 It should be anticipated that, as enzyme technology develops and the search for new enzymes antibiotics continues, an increasing number of enzyme-based transformation will find commercial application. 

The class A enzymes have Mx values around 30,000. Their substrate specificities are quite variable and a large number of enzymes have emerged in response to the selective pressure exerted by the sometimes abusive utilization of antibiotics. Some of these new enzymes are variants of previously known enzymes, with only a limited number of mutations (1 4) but a significantly broadened substrate spectrum while others exhibit significantly different sequences. The first category is exemplified by the numerous TEM variants whose activity can be extended to third and fourth generation cephalosporins and the second by the NMCA and SME enzymes which, in contrast to all other SXXK (3-lactamases, hydrolyse carbapenems with high efficiency. Despite these specificity differences, the tertiary structures of all class A (3-lactamases are nearly superimposable. 

Acetylsalicylic acid irreversibly inhibits both COX-1 and COX-2 by acetylating the enzymes. Since mature platelets lack a nucleus, they are unable to synthesise new enzyme. The anti-platelet effects of acetylsalicylic acid persist therefore throughout the lifetime of the platelet and the half-life of this effect is thus being much longer than the elimination half-life of acetylsalicylic acid (15 min). Since new platelets are continuously launched into the circulation, the clinically relevant anti-platelet effect of aspirin lasts for up to five days. This is the reason why low doses of acetylsalicylic acid (ca. 100 mg per day) are sufficient in the prophylaxis of heart attacks. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Pharmacology. Disulfiram is almost completely absorbed after oral administration. Because it binds irreversibly to ALDH, renewed enzyme activity requires the synthesis of new enzyme. This feature creates the potential for the occurrence of a DER for at least 2 weeks after the last ingestion of disulfiram. Consequently, alcohol should be avoided during this period. 

Following inhibition by methyl parathion, acetylcholinesterase activity recovers as a result of the synthesis of new enzyme, generally at a rate of approximately 1% per day. However, the symptoms of methyl parathion poisoning usually resolve much more rapidly. Therefore, even though they are symptom-free, persons poisoned by methyl parathion may be hypersusceptible to its effects and should avoid reexposure for several weeks (Aaron and Howland 1998 Proctor et al. 1988). 

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