Amino acids active site, /3-galactosidase

Based on the pH optimum for /3-galactosidase and your knowledge of the pAa values of the various amino acid side chains, can you hypothesize which amino acids might be present or important in the active site of the enzyme ... 

The /3-galactosidase study is an excellent example of the power of site-directed mutagenesis. Huber, Miller, and colleagues prepared and examined five Glu-461-/8-galactosidase substitutions (Asp, Gly, Gin, His, and Lys) (136, 139). All substitutions had /teat values less than 0.3% of the wild-type enzyme except the His-461 mutation, which was approximately 6%. For most of the substitutions it was possible to quantify K, /teat. s. and rates of galactosylation and degalac-tosylation for three substrates, and K values for three inhibitors. Different enzyme kinetic properties resulting from different amino acid substitutions confirm that Glu-461 is directly involved in catalysis and contributes to active site structure stability. Heat inactivation at 55°C occurred more rapidly with each amino acid substitution compared to the wild-type enzyme, except for the structurally conservative Gin substitution, which was only moderately affected. 

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