Inhibition of the two-subsite β-d-xylosidase from Selenomonas ruminantium by sugars: Competitive, noncompetitive, double binding, and slow binding modes

Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

“…Equation (4) describes competitive inhibition and it determines the single inhibition constant, K i . Equation (5) describes noncompetitive inhibition, which is needed for D-xylose because in addition to forming the enzyme-Dxylose complex that defines the K i term, it forms the enzyme-D-xylose-4NPX complex [16,18] that defines the K is term of Eq. (5).…”

“…(5). Because the enzyme-D-xylose-4NPA complex does not form [16,18], K i D-xylose values were determined for some of the mutants acting on 4NPA with initial data fit to Eq. (4) simply to affirm that the values are similar to the K i D-xylose values determined for the enzyme acting on 4NPX with the initial-rate data fitted to Eq.…”

“…The remaining activity was fit to Eq. (7) to determine half-activity pH on the acidic side (K a1 0.5 ) and basic side (K a2 0.5 ) a Values from [23] b Triple mutant, T265A-P328L-N516D c Double mutant, W145G-N516D the dissociation constant of 4NPX from the enzyme-Dxylose-4NPX ternary complex, in which the xylosyl moiety of 4NPX is thought to bind to subsite ?1 and D-xylose in subsite -1 [18]. All of the mutations at position 145 cause loss of affinity at subsite ?1 for 4NPX as indicated by the increased K is values of Table 2.…”

“…SXA has the simplest active site possible for an efficient glycoside hydrolase by comprising only two subsites [3], subsite -1 for binding the substrate glycone and subsite ?1 for binding the substrate aglycone. Because the simplicity of SXA lends itself to study, and because of the potential practical importance of SXA, a good deal has been learned about its structure-function properties [3,[16][17][18][19][20][21][22][23].…”

β-d-Xylosidase From Selenomonas ruminantium of Glycoside Hydrolase Family 43

“…1b), SXA in the dianionic form does not bind 4NPA [3]. Thirdly, whereas 4NPX forms a ternary, inhibited complex (SXA-D-xylose-4NPX), 4NPA does not [11]. In this work, pH profiles of steady-state kinetic parameters for SXA-catalyzed hydrolysis of 1,4-β-D-xylobiose (X2) and 1,4-β-D-xylotriose (X3) are determined for comparison with those of 4NPX and 4NPA and analyzing the affinity of catalytically inactive D14 −…”