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Table 3 Results from the simulations and estimates of diffusion times. (a) Parameters used in the simulation: apparent diffusion time τD based on crystal size, initial enzyme concentration E0, outside substrate concentration S0, rate coefficients k1 … k3, and product inhibition Ip (Scheme 1). (b) Occupancy of the various enzyme species as obtained by the simulation. They can be compared to occupancy values listed in Table 2. E free enzyme, ES non-covalently bound ceftriaxone in the active site, with leaving group present, E-CFO* enzyme intermediate with CFO bound covalently, P free product (CFO). (c) Diffusion times τD, and times t to reach stoichiometric concentration in the shard and needle crystals. As an example, estimates for the 1:4 (crystal:CEF) mixing ratio are listed. Time variations between 200 mmol/L and 300 mmol/L CEF are negligible. Times are lower limits, since they are estimated from diffusion in water. Even if they are allowed to vary by orders of magnitude, sufficient occupancy would be achieved after 30 ms. They also imply that the time resolution may be given by the mixing times (Table 1) in some crystal forms, and not by the diffusion times, since the former are longer than the latter

From: Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

(a)
τD (ms) E0 (mmol/L) S0 (mmol/L) k1 (L mmol−1 s−1) k−1 (s−1) k2 (s−1) k3 (s−1) IP (mmol/L)
15/1.5 25 200 3.2 0.01 7 0.8 6
(b)
  0 ms 30 ms* 100 ms 500 ms 2 s  
E (%) 100 10/2 2 1 1  
ES (%) 0 84 58 19 59  
E-CFO* (%) 0 6/14 40 80 40  
P ([mmol/L) 0 0.05 0.5 5.8 23.4  
(c)
  Shard-shaped crystals Needle-shaped crystals  
BlaC concentration in crystal 16 mmol/L 30 mmol/L  
Average size([μm3) 10 × 10 × 3 5 × 2 × 2  
τD 3.5 ms 0.8 ms  
Mixing ratio crystal:CEF 1:4 1:4  
CEF = 200 mmol/L
CEF after mixing 160 mmol/L 160 mmol/L  
Time to reach stoichiometric conc. 0.36 ms 0.17 ms  
CEF = 300 mmol/L    
CEF after mixing 240 mmol/L 240 mmol/L  
Time to reach stoichiometric conc. 0.24 ms 0.10 ms  
  1. *For 15-ms and 1.5-ms diffusion times, respectively