Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate

Abstract

Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions.

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

“…Pyrophosphatase activity was measured at a saturating concentration of PP I (5 mM) and the rate taken to be the k cat . The enzyme was modelled using the one substrate Michaelis‐Menten equation, and using the K M determined in a recent study on this enzyme . The addition of the ttPPiase alleviated the majority of the inhibitory effects seen previously and the CAR reaction went to completion within one hour (Figure B).…”

Self-assembling bubble carriers for oral protein delivery

“…A previous study indicated that SDS has a binding affinity for both free enzymes and the enzyme-substrate binary complex. Proposed molecular mechanisms of proteolytic inhibition by SDS include local conformational changes or altered charge distributions of the proteases induced by their specific binding of SDS [31].…”

Silver Binding to Bacterial Glutaredoxins Observed by NMR

“…The reciprocal values of these were used to produce Dixon plots showing an upward parabolic curve with slopes increasing along with silver concentration (Figure 11d). Dixon curves with parabolic trends have previously been described to explain enzyme inhibition with compounds interacting at multiple binding sites [74,75]. We propose that these trends also can describe inhibition in shared metal complexes: where one atom of silver can inhibit two molecules of GRX, an exponential increase in the rate of inhibition is expected.…”