Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains |
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Authors: | J J?msen A A Baykov R Lahti |
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Institution: | Department of Biochemistry and Food Chemistry, University of Turku, Finland. |
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Abstract: | We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing
the protein with the capacity to be allosterically regulated by adenine nucleotides (J?msen, J., Tuominen, H., Salminen, A.,
Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327–333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated
by diadenosine polyphosphates (AP
n
A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2′(3′)-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding.
The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it
to more rapidly undergo conformational changes in response to binding. |
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