applied Enzymology Asha Manoharan and Joseph H. Dreisbach University of Scranton, Scranton, PA 18510 Conversion of scientific discoveries into technology, that is, the application of the discoveries to industry, medicine, and research, is one of the goals of science. The histom of research in enzymology parailels the history of biochemis&, which originated with investigations of the process of fermentation. Our understanding of the structure and activity of these biological catalysts has advanced to the point where enzymes are now used in numerous applications in a wide variety of areas. Background People have been aware of the chemical chanees catalvzed by enzymes for ages. Louis Pasteur incorrectly ielievedihat processes such as souring of milk and fermentation of carhohydrates to yield a l ~ o h o i r e ~ u i r eliving d cells. In 1878 Willy Kuhn proposed the name "enzyme", meaning "in yeast" in Greek, and in 1897 Edward Buchner showed that enzymes catalyzed sugar fermentation despite the absence of living cells. The first enzyme to be isolated and crystallized, urease, was reported by James Sumner in the early 1900's. Since this significant accomplishment, the science of enzymology developed and flourished to the extent that these molecules now play an important role in industry, medicine, and food science. Enzyme Structure and Reactlvlty One desirable feature of enzymes is their specificity for one or a few substrates. This specificity is due t o the arrangement of specific amino acid side chains a t the active site. These molecular groups participate in the binding of the substrate and its subsequent conversion to product. The active site occupies a relatively small part of the enzyme surface, and substrate binding involves weak, noncovalent interactions. The molecular groups include polar and nonpolar amino acid residues, which create hydrophobic and hydro~hilicmicroenvironments. Thus. enzvme activitv depends not only on spatial arrangements of binding and-catalvtic p r o. u ~ sbut also on the environment in which these . . groups exist. Enzyme suhstrate specificity is often explained in terms of the "lock-and-key" hypothesis. Although this is an excellent model system to describe enzyme action, i t ignores the flexibility inherent in globular proteins. Koshland presented a more sophisticated model in 1958 that he refers to as an "induced fit" model. That is, the enzyme is required to undergo subtle conformational changes in response to suhstrate binding. One can envision molecules similar in structure to the enzyme suhstrate that are less capable of introducing the required conformational chanee in the enzvme. ~ l t h o & hso& enzyme catalysis might occur, reaction iates would be lower than with the actual substrate molecule. This affinity of the enzyme for various reactants is de-
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Journal of Chemical Education
Km [substrate] Figure 1 lnittal reaction velocity so s funmon ol substrate cancsmratlan M, Is me subshate concentration requ rea to ream one-hall max mum relacdy
scribed quantitatively using the Michaelis-Menton constant (K,). The mathematical meaning of K, depends on the mechanism of the particular enzyme-catalyzed reaction and can be complicated. A simple interpretation defines K, as the substrate concentration required to reach half-maximum velocity for the reaction (Fig. 1).Kmis independent of enzyme concentration and, in some cases, is a measure of the affinity of an enzyme for a given suhstrate. The Michaelis constant has dimensions of concentration, typically moles per liter. TWO enzymes are capable of catalyzing the phosphorylation of glucose using ATP as phosphate donor (eq 1).
Hexokinase has a very low K, for glucose (37 pM while glucokinase has a much higher value (10 mM) ( I ) . Hexokinase has a much greater affinity for glucose than does glucokinase. When selectine an enzvme for a technical awwlication, i t is .. important to consider a number of factors including specificitv. Cost effectiveness is a maior factor in evaluating the fiasibility of a specific enzyme application. The enzyme must be reasonably stable to the pH, temperature, and ionicstrength conditions used in the process. Also important is the availability and cost of the enzyme.
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Enzymes as Therapeutic Agents
The enzyme asparaginase catalyzes the conversion of the amino acid asparagine into aspartic acid and ammonia (eq 2). has been used in the treatment of certain . Asoaraeinase . types of cancer (2). I t has been shown that tumor cells that are sensitive to aswaraeinase therawv have insufficient asparagine synthetase activity (eq 3 j i n d are dependent on exogenous asparagine for growth and survival. For these sensitive tumors, asparagine is an essential amino acid and, when depleted by reaction with asparaginase, these sensitive tumor cells are killed (3). Resistant tumors possess much higher levels of asparagine synthetase than normal tissue and thus are capable of producing sufficient asparagine for tumor growth even in the presence of asparaginase (2). Reaction of asnaraeinase with the oatient's immune svstem also reduces t i e e6ectiveness of t i e therapy, and attempts have been made to utilize asoaraeinase from other sources when . immune sensitivity develops (4).
dure. Numerous genetic diseases have been identified and although the molecular error is traced to a defective gene, this type of therapy is focused on the gene p r o d u c t t h e defective enzyme molecule. Gaucher's disease is one example of a hereditary disorder. Among its symptoms are marked enlargement of the spleen and liver, and mental retardation is observed in the infantile form ( f i ~ It. has been demonstrated that patients suffering from this disease accumulate a glywlipid, glucocerebruside, in these organs. Normally thih lipid is hydrolyzed by 2glucosidase into ceramide and glucose (eq 41. These products are further catabolized in a varietv of metabolic reactions. 3Glucosidase, which is normally stored in the lysozomes, is not active in ~ a t i e n t with s Gaucher's disease. Some success has been obse&ed in experiments that administer 8-glucosidase to patients (5).
...
HC=CH-(CHArCHs 0-g1uc.m
n cmcooI II l HIN--C--CHrCH+ HIO -t -0OC-CHpCH + 1 INHp N Ha' L-asparwine
1 I
HIN-C--CHrCH
(2)
I
NHs+
L-.sparate
0
NHs Ammonia
L-srparatl
I NHs' L-slutmine
cooI I
L-aaparagioe
requirement todirect the enzyme to the specific target tissue and to subcellular organelles. One approach is to bind the enzvme to a cell-swecific receotor molecule that allows that celito internalize the complex. Another method is to encapsulate the enzvme in some tvoe of vesicle such as a liwosome or erythrocyte (5). Many other lysosomal storage diseases such as Tay-Sachs and Fabry's disease are also under study for enzyme replacement therapy. Enzymes Used in Diagnosis
cooI I
+ -0CCCHr C H r C H
NHs+
anmidc
A major difficulty in enzyme replacement therapy is the
+ AMP + PP
(81
NHI+ L-gIutam.tc
Although asparaginases are found in many sources such as bacteria, plants, and vertebrates, not all asparaginases are clinically useful. Those asparaginases have little or no activity against tumors generally have higher K , values or react rapidly with the patient's immune system (2). Unfortunatelv. as is the case with manv theraoies involving chemicals, side effects are noted with ¶$nase treatments. As with other situations. research to reduce these side effects continues, and risk-bknefit relationships are examined prior to treatment. The success with asparaginase therapy of certain tumors has generated interest in other amino acid degrading enzymes and their potential as antitumor agents. Glutaminase, cysteine desulfhydrase, and a number of other enzymes are being examined for their effectiveness as therapeutic enzymes ( 3 , 4 ) . Enzyme Replacement
Another theradeutic anolication of enzvmes. . . also verv much in the expe&nentaistage, is the replacement of defective enzvme activitv hv addition of an active enzvme ohtained 6om another source (5). The concept is similar to hormone replacement therapy, which is a common proce-
Enzymes are used in two different ways in the clinical laboratory. Measurement of enzyme levels in serum provides much information on the nature and extent of tissue damage or activity. This aspect of clinical chemistry has become important since the elucidation of the roles of specific enzvmes in the metabolic nathwavs and the develooment of rapid and reliable enzyme ass& procedures (7). k second wav of usina enzvmes in the clinical laboratorv is as reagents in rapid an2 sensitive analytical procedures. Enzymes in Heart Disease Monitoring serum levels of certain enzymes provides information on the extent of myocardial infarction. Enzymes are released from the damaged heart tissue into plasma at different times after onset of necrosis, and, since the half-life of these enzymes in plasma differs, the rates of disappearance of the enzvme activitv in serum are also different. Three enzymes are routinel; monitored when heart disease is suspect. They are creatine kinase (CK), glutamic oxalacetic transaminase (GOT), and lactate dehydrogenase (LDH) (8).The maanitude of the increase is a rouah indication of the extent of damage and the length of time the levels are increased is an indication of continuing necrosis. Enzymes a s Tools in Clinical Analysis Blood glucose determination is one of the most widely employed clinical analyses. The glucose tolerance test is a common diagnostic procedure used to evaluate a patient's ability to metabolize glucose. A person is provided with a Volume 65
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glucose solution to be taken orally and blood glucose concentration is monitored for up to four hours. Normally blood glucose levels rise for the first hour and then quickly return tooriginallevels, about 80 mgof glucose per 100 mL of blood. A patient suffering from diabetes mellitus does not metabolize glucose normally, and blood glucose levels increase dramatically above normal ranges during the course of the test. A number of different enzymatic methods for glucose analysis are available. In one method two enzyme-catalyzed reactions are coupled. Glucose is first phospborylated with bexokinase (eq 1) followed by oxidation with glucose-6phosphate dehydrogenase (eq 5).
Table 1.
Some lndustrlal Uses of Mlcroorganlsms
process or ~ ~ c d u c t
Microbe
citric acid gramicidin chloramphenicol isopropanoi and +butanoi 6-aminapeniciiianic acid acetic acid biodegradation of petroleum cheese production
ethanol production
Table 2.
The stoichiometry of the reactions is such that for every mole of D-glucose present one mole of nicotinamide adenine dinucleotide phosphate (NADPf) becomes reduced. Since NADPH absorbs light a t 340 nm but the oxidized form does not, one can determine the amount of glucose present by measuring the change in absorbance a t this wavelength. Enzvmic methods of auantitative analvsis are advantageous because the methods are specific, rapid, and requireonly verv small amounts of sample. Enzvmatic methods for analysisof urea, cholesterol, chblesteroiesters, triglycerides, and numerous otber metabolites are routinely available to clinical laboratories. lndustrlal Applicatlon of Enzymes The efficiency and specificity of enzyme catalysts render them excellent for use as industrial catalvsts. Since thev are active under mild reaction conditions and produce no-side products, costs for the process can often be greatly reduced. In some cases, especially in those processes that require more than one reaction to go from reactants to final produrtr, whole cells are used rather than isolating and purkying individual enzymes. Use of whole cells reduces the expense of enzyme puhfication, but side reactions do occur. These side reactions sometimes are necessary to insure the quality of the product. Brewing and cheesemaking are some of the oldest industrial processes utilizing living organisms. The major end products, alcohol in brewing and various organic acids and sugars in cheesemaking, are produced by the orzanisms. hut the smaller ouantities of other metabolites also produced are responsible for imparting the subtle flavors characteristicof various beers. wines. and cheeses. Microbial fermentations are also used in the p;oduction of a variety of organic comoounds. Cells are selected or engineered, result. ing in an enhanced metabolic capability to produce certain desired com~ounds.Table 1lists some microbial processes currently uskd in industry. Advances in genetic engineering and gene cloning have made it nossible to develoo strains of microbes capable of produciig high levels of prdducts. Some of these compounds are oroducts of eenes from other organisms that have been spliEed into themicrobe. Insulin and human growth hormone are proteins currentlv produced using genetically al.. tered microbes.
Aspergilius niger Bacillus brevis Streptomyces venezuelae Clostridiumbmricum E. COC strains Clostridium thermoaceticum Candida sp. and many others L a ~ t ~ b a c i lsp.: l~s Streptococcus sp.; Penicilliumsp.: and ProprionibactWm sp. Saccharomyces cadbergensis
Some Enzvmes Used In Commercial Aoolications
Enzyme
Source
Application
aspraginase w-~mvlase p101ea~es coliagenases am noacy asst glucaamyiasa glucose isomerase
E. coii B, stearoihmoohNus Aspergillus niger Closadum histoIy?icum Arpergillullus o'yzae Aspergillus nigw Bacillus coagulans
antitumor therapy bakino. brewino meat process ng own lreatmenl bemino acia pr0a.clion high-gi~cosesyrup high-fructose syrup
Isolated Enzymes in Industrial Processes Enzymes are used in a number of different ways in the food industry. Carbohydrates required for fermentation and carbon dioxide production are derived from the hydrolysis of starch by amylases. Deficiencies of amylase can occur when wheat is mechanically harvested and germination does not occur properly. Addition of a-amylase results in sustained maltose production that can he fermented by yeasts (9). Commercially available meat tenderizers contain a mixture of proteolytic enzymes with various specificities. These proteases hydrolyze the peptide bonds of connective tissue protein (collagen and elastin) to yield smaller peptides. The most commonly used proteases are trypsin, chymotrypsin, papain, and some otber microbial proteases (7). The commercial market for sweetening agents is very large. Enzymes are used to process starch into a mixture of glucose and fructose. Since the ratio of sweetness of glucose&crose:frnctose is 1:1.5:2.5 a elucose-fructose mixture produced from cornstarch is a more cost-effective sweetner than
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Journal of Chemical Education
Figure 2. Methods for immobilizing enzymes. A. Enzyme is entrapped in a cross-linked polymer. 0. Enzyme is covalently banded to an insoluble support.
processed sucrose (11). Table 2 lists some enzymes and sources t h a t have found extensive use in the food industry.
result of immobilization, the enhanced stability and reusabilitv renders the process advantageous from a practical standpoint.
lmmobillzlng Enzymes Some of the major concerns in applied enzymology are the cost and stability of the enzyme catalysts. By linking the enzyme t o some solid support, one facilitates recycling of t h e catalvst and also, in many cases, enhances its stability. Some indudtrial enzymes and whole cells have been immobilized and are currently in use (12). There are a number of methods for immobilizing enzymes, b u t they can all be classified into two general categories: physical entrapment and chemical attachment (Fig. 2 ) . Entrapment involves the formation of a cross-linked polymer such a s starch or . oolvacrvlamide eels around the enzvme. " T h e enzyme is trapped in the matrix where lower molecular weieht substrate and oroduct molecules can diffuse freely i n t i and out of the matrix. Chemical attachment of the enzyme to a n insoluble support is performed in a variety of ways, and many insoluble materials such a s glass, polysaccharides, and collaaen can act a s supports. T h e enzyme is bound to the support by reaction of a chemical group not required for catalysis. Although some activity is lost as a
Conclusion There exists a large number of chemical and industrial applications of enzymes, b u t this article describes only a few examples. Recent advances in isolating, purifying, and immobilizine enzvmes and in microbial technoloev indicate t h a t applied enzymology is a n important factor scientific and technological development. Literature Cited 1. Ferdinand, W. T h s E ~ y m Molecule: e Wiley: New York, 1976;p 139. 2. Capizei,R. L.;Cheng,Y-C. InEnzyme8osDrugs. Ho1eenberg.J. S.;Roherts J.,Eds.: Wilev: New Yort. 1981: Chaoter 1.o 1.
.. yo& 1975;Chspfer 11. p301.
10. OIS~",A.c.:Korus. R. A. lnEnrymoa inFoado"dBe".rog. Proeesring,0ly.R.L.: St. Ange1o.A. J. Eds.: American Chemical Society: Washington, DC, 1977;Chapter 7, p 7-
&-.
11. MaeAllister, R. V.:Wardrip, E. K.; Schnyder, B. J. In Ref 9, Chapter 12, p 332.
Journal of Chemical Education: Software Under agrant from the Dreyfus Foundation, Project SERAPHIM and the Journal of Chemical Education have been collaborating to design and produce an electronic journal that will provide software authors with the same prestige and academic credit afforded authors of written journal articles. Editor Uni Susskind and SERAPHIM Director John Moore are putting finishing touches on the first issue (Vol. I, No. 1)of The Journal of Chemical Education: Software, sponsored by the Dreyfus Foundation. This will be a peer-reviewed, officially abstracted publication of the Journal that will contain instructional computer programs on floppy diskettes. The first issue, prepared for Apple I1 computers, is oriented toward a teacher who wants to demonstrate chemical principles to an entire class using just one computer. Its contents will include: "a-Scatter", by Robert Rittenhouse, which illustrates on a microscopic level the scattering of a-particles by atomic nuclei. "Aufbau", by Dale Jensen, which shows graphically the build-up of electron configurations hoth on an energy-level diagram and on a periodic tahle. "Periodic'I'ableWorks", by Jon Holmes, which converts the Apple I1 into an elertnm:c prriod~ctahle on which a teacher can highlight demems, groups, or periods, display periodic trends. wew data. ~ R e m r m h those ~ r illuminated periodic tablesin "niversity Lecture halls? But much better!) "Wave", by James Hutchison and Martin Rose, and "Sum of Two Waves", by Robert H. Good, which illustrate wavelength-frequency-velocity relationships and wave interference and combination. Several future issues of JCE: Software are now being planned. "SERAPHIM Laboratory Manual" by Daniel Krause, for Apple 11, will contain updated directions for constructing interfaces, carefully selected and written experiment handouts for students, and a new, easy-to-use, data-collection program to control the interfaces. "One-Computer Classroom: Gas Laws" edited hy Uni Susskind, will provide several Apple classroom demo programs involving gases. "Spreadsheet Templates for Physical Chemistry" by David Whisnant, for hoth Apple and MS-DOS machines, will include "what if?" exercises, student guides forchapter-end problems in a major textbook, and an introductory set of directions for students to learn spreadsheets. "HPLC", by Rohert Rittenhouse, and "KC? Discoverer", by Aw Feng, John Moore, William Harwood, and Robert Gsyhart, for MS-DOS machines, are expanded versions of programs already in the
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If you are interestrd in purchasing the first electronic issue, or if you are interesfrd in n suI,scription for yourself or your lihrary, pleasr wrm to: John W. Moore, D~rertor,Project SERAPHIM, Department of Chemistry. Eastern .Michigan university; ~ ~ s i l a n MI t i , 48197 for more information.
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