Table 2

Structure-function correlations of various adenylyl cyclase mutants. Mutations made in different isoforms of adenylyl cyclase (first column) are mapped onto VC1 or IIC2 (second column). [Mutation name specifies the residue at the given position, mutated to the residue listed at the end (50).] Their observed phenotype is described in the third and fourth columns, and their role in the VC1·IIC2·Gs αcomplex is described in the fifth column. EC50, median effective concentration; IC50, median inhibitory concentration.

Mutation (isoform)Context(VC1 or IIC2)VmaxFsk*/ VmaxG*/% G boundEC50 G*/ IC50P-site§/ Km ATP*Structural correlateRef.
Wild type++/++/++++/++/++
F293A (ACI)F379 (VC1)++/+/ND−/ND/NDInteracts with Gs α; C1·C2interface (27)
D338A (ACI)D424 (VC1)–/+/+NC/ND/NDProtein fold; C1·C2 interface (10)
R348A (ACI)R343 (VC1)++/++/++++/+/++Salt bridge to D424(10)
K350A (ACI)K436 (VC1)++/++/+++/+/++C1·C2 interface(10)
D354A (ACI)D440 (VC1)–/–/+NC/ND/NDCoordinates Mg2+; potential base(10)
R398A (ACI)R484 (VC1)+/–/++ NC/+/++Binds Pγ of pyrophosphate (10)
H402A (ACI)H488 (VC1)–/–/–NC/ND/NDProtein fold(10)
D419A (ACI) D505 (VC1)–/–/++ NC/ND/NDSalt bridge to K436(10)
E432A (ACI)| E518A (VC1)| E518 (VC1)++/+/++NC/+/+Salt bridge to R484(10, 12)
R913A, L914A N916A, E917A D921A (IIC2)Same++/–/ND–/ND/NDInteracts/supports contacts with Gs α in α2 helix; C1·C2interface(27)
D908A (ACI) D923 (IIC2)–/–/–NC/ND/ND Protein fold; C1·C2 interface(10)
K921A (ACI)K936 (IIC2)+/+/++NC/+/++Salt bridge to D923; C1·C2 interface(10)
K923A (ACI)K938 (IIC2)+/+/++NC/–/+Binds adenine N1; C1·C2 interface(10)
H950A (ACI)H968 (IIC2)–/–/+NC/ND/NDProtein fold; strained side chain(10)
N987A, H989A F991A, N992A (IIC2)Same++/+/ND++/ND/NDInteracts/supports contacts with Gs α in α3 helix(27)
R979A (ACI)R997 (IIC2)–/–/–NC/ND/NDProtein fold; salt bridge to D1031(10)
Y999A (ACI)Y1017 (IIC2) –/–/+NC/ND/NDProtein fold(10)
D1000A (ACI)D1018 (IIC2)–/–/+NC/ND/NDBinds adenine N6(10)
N1025A (IIC2)Same–/–/NDND/ND/NDActive site structure; water coordination(33)
R1029A (IIC2) R1011A (ACI)R1029 (IIC2)–/–/++NC/–/++Stabilizes transition state; binds Pβ of pyrophosphate in P-site inhibited complex(10, 33)
Y1054A (IIC2)SameND/–/NDND/ND/+Protein fold(12)
R1059A (IIC2)SameND/++/NDND/ND/+ Protein fold(12)
K1065A (IIC2)SameND/–/NDND/ND/+ Binds Pγ of pyrophosphate(12)
K1049A (ACI)K1067 (IIC2) ++/++/++++/+/++Binds N1022 in P-site complex(10)
  • * Symbols: (++), wild-type value; (+), 2- to 10-fold decrease in activity or K m; (–), ≥10-fold decrease or inactive; ND, not determined; NC, not calculated because affinity was too weak or negligible activity. Activities correspond to those measured with Mg2+ as the metal ion.

  • ACI forskolin-stimulated activity was measured with Mn2+ as the metal ion.

  • Symbols: (++), ≥50% of wild-type binding; (+), 20 to 50% of wild-type binding; (–), ≤ 20% of wild-type binding or no detectable binding.

  • § Symbols: (++), wild-typeK i; (+), 5- to 100-fold increase inK i; (–), >100-fold increase inK i.

  • | Activity is dependent on Mn2+.