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Towards Rational Design of High-efficiency Enzyme Cascade Yifei Zhang, and Henry Hess ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01766 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017
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ACS Catalysis
Towards Rational Design of High-efficiency Enzyme Cascade Yifei Zhang, Henry Hess* Department of Biomedical Engineering, Columbia University, New York, NY 10027, United States KEYWORDS : enzyme cascade, coupled kinetics, proximity channeling, enzyme on scaffolds, co-immobilization, compartmentalization, activity enhancement
1. Introduction The vision of assembling molecules in a manner similar to a factory, where parts pass from workstation to workstation, is highly appealing. A key requirement to achieve such control over multistep reactions is the ability to control the transport of intermediates from reaction site to reaction site. Biology utilizes compartmentalization, metabolons, and scaffolds to direct the transfer of intermediates between enzymes catalyzing a reaction cascade. Frequently, “channeling” of the intermediate from one active site to another is achieved via tunnels or interfaces. Recent advances in nanotechnology have enabled the design of biomimetic architectures to spatially organize enzymes on DNA or protein scaffolds with nanometer precision, and an enhanced production has been observed, which is thought to arise from accelerated transport of the intermediate between enzymes in close vicinity (proximity channeling).1-4 More and more sophisticated architectures such as nanocaging,5 clustering6 and even bridging the cascade enzymes7 have now emerged to facilitate the transfer of intermediates in artificial systems. However, the mechanism leading to accelerated product formation is still widely debated. Wheeldon et al. have summarized the biological concept of substrate channeling comprehensively and described some examples of enzyme cascades on artificial scaffolds in two outstanding reviews.8.9 Other recent reviews were focused on the architectures of the cascade enzyme systems and more or less accepted the hypothesis of proximity channeling.10-14 Here, we reiterate our point of view (first expressed in 2012)15-18 that the enhancements are not caused by the proximity of the enzymes and review the recent evidence supporting this perspective. In particular, we highlight evidence that the scaffold alters the enzyme characteristics. Finally, we summarize recent insights and the resulting viable strategies to achieve highly efficient multienzyme cascade reactions.
2. Proximity does not enhance the overall activity of an enzyme cascade 2.1. Substrate channeling in nature uses confinement not proximity. Natural substrate channeling was observed in several bifunctional enzymes and multienzyme complexes (e.g. tryptophan synthase,19,20 carbamoyl-phosphate synthetase,21 thymidylate synthase–dihydrofolate reductase22). These structures have physical tunnels which can guide the flux of intermediate molecules to the next active sites through hydrophobic/hydrophilic or electrostatic interactions. In some cases, the intermediate molecules can also be efficiently transferred by a swinging protein domain23 or dynamically formed interfaces between a transient heterodimer.24 The physical or chemical confinement prevents the intermediate from escaping to the bulk solution, thereby creating a high local concentration of intermediate which increases the throughput. The efficiency of tunneling is further improved by a synchronization of the catalytic cycle of the involved enzymes, which has been shown to occur in nature via allosteric communication.25 Although confinement often places the involved domains in the proximity of each other (e.g. the discovered molecular tunnels are in the range of 2 to 10 nm), the key is that the escape of the intermediates to the bulk is prevented. For all substrate channeling mechanisms, the steady state rate of product formation in a solution containing only the enzymes and the substrates is not affected if the individual kinetics of cascade enzymes is unchanged as we discuss in detail in section 2.2.26-28 However, substrate channeling is beneficial in the complex intra- and extracellular environment, where escaping intermediates can be lost to the environment and competing reactions.
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Figure 1. Stylized bucket brigade to illustrate cascade kinetics. Workers transport water to a reservoir (a) via an intermediate reservoir, where the flux from the tap depends on the water level in the intermediate reservoir; (b) with a rope, in which the time cost for "channeling" is negligible; (c) with a rope and an intermediate reservoir, but a leaky bucket; (d) via an intermediate reservoir and more workers carrying water from the intermediate reservoir to the second reservoir. The time course of the filling process changes and permits conclusions regarding the process, but different mechanisms can have a similar time dependence.
2.2. Kinetics of sequential and coenzyme regenerating cascade in the absence of channeling Sequential cascade. The kinetics of a two-enzyme cascade can be illustrated by a stylized bucket brigade (Figure 1). When two workers transport water through an intermediate reservoir, the initial flux from the tap depends on the water level in the intermediate reservoir, but the final velocity is limited by the slower worker (we assume here it is the first one). The water volume in the second pool increases as the red curve in Figure 1a. If the workers can pass buckets directly between each other using a rope, the transport velocity is constant and the water volume increases linearly with time (green curve in Figure 1b). When using both rope and reservoir but a leaky bucket, the time course of filling is in between the red and green curve (Figure 1c). However, if there is no rope, but many workers using many taps, the water can be transported to the pool as soon as it is poured in the intermediate reservoir (Figure 1d). Thus, the time course of filling (green dashed line) resembles the one of perfect channeling. The same logic applies to sequential enzymatic reactions. Consider a simple model of an enzyme cascade with freely diffusing enzymes (Figure 2), in which enzyme 1 produces the intermediate substrate (I) at a constant velocity of V1 , and enzyme 2 follows Michaelis-Menten kinetics (Vmax,2 and Km,2, here we set Km,2 equal to 5 μM). The first assumption is valid when the concentration of substrate 1 does not change significantly during the cascade reactions. This situation corresponds to Figure 1a, where the increasing concentration of intermediate accelerates the work of enzyme 2. The whole process can be described with a system of equations under the assump-
tion that the intermediate concentration equilibrates instantaneously in the reaction volume.
r1 V1 r2
(1)
Vmax,2 [ I ]t [ I ]t K m,2
(2)
t
[ I ]t r1t r2 dt 0
(3)
t
[ P] r2 dt 0
(4)
Figure 2 shows the coupled kinetics of a sequential cascade with varying reaction rates of V1 and Vmax,2. The final activity of all reactions will converge to the rate of the slower enzyme. Only when V1
