Aktywność katalityczna pojedynczych cząsteczek enzymów

• Autorzy: Jakubaszek J. , Molski A.

Warianty tytułu

EN

Catalytic activity of single enzyme molecules

• Języki publikacji: PL

Abstrakty

EN

As early as in 1961, it has been demonstrated that turnovers of individual enzyme molecules can be detected [1]. In the nineties, advances in single-molecule methods, in particular in confocal microscopy (Fig. 1), made it possible to monitor more closely enzymatic turnovers at a single-molecule level [2-5]. This led to the discoveries of static disorder and dynamic disorder, and closely related memory effects in enzymatic turnovers [6-8]. Differences in activity of individual molecules of the same enzyme are called static disorder. Time-dependent fluctuations of enzymatic activity are called dynamic disorder. One manifestation of dynamic disorder is the fact that consecutive enzymatic turnovers are not statistically independent, which is called "memory effect". It is believed that static dis-order and dynamic disorder are related to conformational dynamics of enzyme molecules. In this review we discuss current issues of single-molecule enzymology, in particular kinetic effects that are specific to single-enzyme measurements. First we review the conceptual basis of single-enzyme kinetics and the initial work on single enzymes. We focus on the ping-pong mechanism of bisubstrate enzyme reactions (Eqs. (1) and (2)), and consider fluorescence trajectories (Figs. 2 and 3) associated with enzymatic turnovers. Two cases are distinguished. In the first, fluorescence comes from an enzyme molecule and fluorescence intensity jumps called blinking carry information on enzymatic activity. Jumps between a fluorescent (on) and non-fluorescent (off) states (Eqs. (4), (5), and Fig. 2) indicate the moments when the photophysical state of an enzyme changes during enzymatic turnovers. In the second case, fluorescence comes from product molecules whereas enzyme and substrate are non-fluorescent. Fluorescence bursts on a trajectory indicate the moments when non-fluorescent substrate molecules are converted into fluorescent product molecules that subsequently diffuse away from the detection volume (Eqs. (8), (9) and Fig. 3). In Section 1 we present selected experiments implying the effect of conformational dynamics on enzymatic kinetics. In Section 1.1, we discuss cholesterol oxydase and dihydroorotate dihydrogenase as examples of enzymes whose on-off fluorescence blinking is caused by chemical reactions at the enzyme active site. In Section 1.2 we discuss ?-lactosidase and lipase B as enzymes which turnovers can generate fluorescent products from suitably chosen non-fluorescent substrates. In Section 2, we review modeling and simulations of single-enzyme data. The aim of data modeling is to gain insight into single-enzyme activity through analysis of models of increasing complexity. Phenomenological models attempt to capture the essence of single-enzyme kinetics without going into molecular details. If a model is simple enough it may allow analytical solutions. For instance, a simple model of on-off blinking is given in Scheme (25). This scheme is capable of reproducing memory effect that can be visualized by a two-dimensional histogram of consecutive on and off times (Figs. 5a and 5b). Finally, in the last section we present an outlook for single-molecule enzymology.

Słowa kluczowe

PL

kinetyka enzymatyczna; pojedyncze cząsteczki; spektroskopia fluorescencyjna

EN

enzymatic kinetics; single molecules; fluorescence spectroscopy

• Wydawca: Polskie Towarzystwo Chemiczne
• Czasopismo: Wiadomości Chemiczne
• Rocznik: 2008
• Tom: [Z] 62, 7-8
• Strony: 635--658
• Opis fizyczny: bibliogr. 27 poz., wykr.

Twórcy

autor

Jakubaszek J.

autor

Molski A.

• Pracownia Dynamiki Procesów Fizykochemicznych, Wydział Chemii UAM, ul. Grunwaldzka 6, 60-780 Poznań

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