Carbonic Anhydrase Catalysis An Experiment on Enzyme Kinetics Greg T. Spyridis a n d J. E. Meanyi Seattle University, Seattle, WA 98122 Y. Pocker University of Washington, Seattle, WA 98195
Undergraduate enzyme kinetic experiments can be a valuable learning experience for students hut can also be a great source of frustration. For example, many enzymes in solution denature rapidly, and for enzymes available as ammonium sulfate suspensions it is often difficult to prepare solutions of known specific activity that give reproducible results from one series of kinetic runs to the next. We have developed an experiment on enzyme kinetics which is virtually free of surh prohlrms. Thc experiment utilizes hovine ervthrocyte carbbnic auhydrase (BCA), a very stable enzyme commercially available in lyophilized form. The white powder can be weighed precisely to produce stable solutions of k n o w molarity. Best of all is the fact that carbonic anhydrase lends itself well to a wide variety of kinetic studies typical of those used in the elucidation of enzyme mechanisms. For example, determinations of pH-rate profiles and solvent deuterium isotope effects, as well as inhibition and catalytic versability studies, can be carried out ~reciselveven usina- simple . experime&al methods. Thus, a'fter preiiminary specific activity determinations, each student is able to develop his or her own laboratory project. Carbonic anhydrase is most readily isolated from erythrocytes where it is present in about 0.1% concentration. The enzyme catalyzes the reversible hydration of carbon dioxide
After C02 is formed as a product of metabolism, it is hydrated and transported through the blood as bicarbonate ion. At the interface of the lung, carbonic anhydrase rapidly reconverts bicarbonate ion back to COz, which is exhaled. At one time it was believed that carbonic anhydmsv was .~, ( I ). Howver. specific to a single set of i u h s t r ~ t ~COJH(:O.; the ciltalytic versatilitv of the imzyme was demonstrated in 1965 by the observation that the enzyme also catalyzes the hydration of aldehydes and the hydrolysis of esters (2). Kinetic studies have revealed many details concerning the mechanism of action of bovine carbonic anhydrase (2-4). As with most enzymes, the activity of BCA is highly pH-dependent. Hydrase and esterase activities generally exhibit a sigmoidal DH-rate profile with an inflection woint around neutrality and optimal activity at pH raliwi alr~vt:8. Such studies indicate tho rntalytic i n \ d m n m t of >inionrzahle group with npK.-7, the basiciorm ofwhirh iscatalytically active 12 ?). HCA is a zinc metalloenzwne in which the sincle zinc ion rwr molecule of the enzyme serves as an obligatory cumpun~mtof catalvsis (2-41. There is evidence suuestinr that sult'unamides inhibit BCA by coordination a t or near thk zinc ion and, further, that it is a zinc bound hvdroxidelwater which is trans-
' Author to whom correspondence should be addressed. 1124
Journal of Chemical Education
ferred in the turnover sten (2-5). For the h k h activitv "isozvme . present in bovine erythroches; it has also lbeen hypothesized that the imidazole group of a histidine residue serves as a general base in promoting the transfer of water from zinc to the electrophilic site on the substrate (3). In this experiment the enzyme-catalyzed hydrolysis of p nitrophenyl acetate (PNPA) is studied usina.a spectropho. tometric method 0
11
BCA
PNPA
The formation of p-nitrophenoxide results in an inrrease in absorbance at 410 nm. T h r rxweriment can be carried our comfortably in one laboratory period. Experimental Equipment Spectrophotometer. Bauseh and Lomb Spectronic 20, Turner Model 330, or similar instrument with the capacity to detect the ahsorbance of light at 410 nm. The total volume for all runs in the enperiment is 5.0 ml. If 3.0-ml euvettes are used, volumes of all solutions must be reduced orooortionatelv. . . Constom lcnaptwr~rn,h o r h All rolutions must be preinculvated at room trnqmilture priur I
