title: Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II.
abstract: Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme. The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid. The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143. All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid. Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold). KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical. The functional data in conjunction with X-ray crystallographic studies of four of the mutants [Alexander et al., 1991 (following paper in this issue)] allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association. The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react. The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide. This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
authors.forename: T L
authors.initials: TL
authors.lastname: Calderone
authors.forename: J F
authors.initials: JF
authors.lastname: Krebs
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D002245:Carbon Dioxide / Q000737:chemistry; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry; D005822:Genetic Vectors; D006878:Hydroxides; D017665:Hydroxyl Radical; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D013329:Structure-Activity Relationship; D014867:Water; D015032:Zinc
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D006878:Hydroxides; D011994:Recombinant Proteins; D014867:Water; D002245:Carbon Dioxide; D017665:Hydroxyl Radical; D004950:Esterases; D002256:Carbonic Anhydrases; D015032:Zinc