Journal of Sol-Gel Science and Technology 15, 57–61 (1999) c  1999 Kluwer Academic Publishers. Manufactured in The Netherlands. Allosteric Regulation of Enzymatic Reactions in a Transparent Inorganic Sol-Gel Material NICOLA H ¨ USING, EMIL REISLER AND JEFFREY I. ZINK Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095 Received August 25, 1998; Accepted November 23, 1999 Abstract. Glutamate dehydrogenase is encapsulated in a transparent porous silicate matrix by using sol-gel techniques. The inorganic polymer is formed around the enzyme (MW > 300,000 D). The enzyme is active in the material, catalyzes the reaction of L-glutamate to 2-oxoglutarate and follows Michaelis-Menten kinetics. The allosteric regulators ADP and GTP inhibit or activate the reaction; at pH 6, GTP acts as a strong activator and ADP acts as an inhibitor. This system involves a complex series of interactions; the co-enzyme NAD + is required for catalysis, large-scale conformational changes accompany the binding of the substrate and coenzyme to the enzyme, the activators/inhibitors must bind to the enzyme to regulate the reactions, and the substrates and products must diffuse through the matrix to and from the binding site. The influence of the unique matrix on the complex enzymatic system is discussed. Keywords: glutamate dehydrogenase, allosteric regulators, sol-gel, enzyme activity Sol-gel procedures can be used to encapsulate enzymes with retention of activity in optically transparent sili- cate glass [1–17]. These new materials are of inter- est for their applications as optically-based biosensors. The porosity of sol-gel glasses allows small molecules to diffuse into the matrix while the large enzymes re- main physically trapped in the pores of the glass. The transparency of the matrix makes it feasible to monitor spectroscopically the reactions that occur in the pores of the glass. The diversity of the enzymatic reactions that are pos- sible in the pores of the silica matrix has not been ex- plored; most of the studies to date have focused on relatively low molecular weight and robust enzymes that were chosen for sensor applications. The effects of the confinement in the pores on the enzymes are vir- tually unknown. One method of exploring such effects is the use of a multi subunit allosteric enzyme such as glutamate dehydrogenase (GDHase) [18] where the in- hibition or activation by ADP and GTP have been well characterized in solution [19] and the conformational changes in the protein involved in its catalytic action are structurally resolved [20]. This system involves a complex series of interactions; the co-enzyme NAD + is required for catalysis, the activators/inhibitors must bind to the enzyme to regulate the activities, the sub- strates and products must diffuse through the ma- trix to and from the binding site, and importantly, large structural changes occur in the enzyme during its function. These complex processes should be influ- enced by the confinement in the matrix and could be inhibited. In this paper we show that allosteric regulators function in a GDHase-doped sol-gel matrix. GDHase catalyzes the oxidation of L-glutamic acid to alpha- ketoglutarate via the imino derivative, the bound coen- zyme NAD being reduced to NADH in this process. L-glutamate + NAD + + H 2 O GDHase ⇀ ↽ 2-Oxoglutarate + NADH + NH + 4 (1) Bovine glutamate dehydrogenase consists of six polypeptide chains [18, 21–25]. The six subunits form the smallest stable active structure with a molecu- lar weight of 330,000. Activity measurements in the