Binding of allosamidin to the three family 18 chitinases of Serratia marcescens has been studied using isothermal titration calorimetry (ITC). Interestingly, the thermodynamic signatures of allosamidin binding were different for all three chitinases. At pH 6.0, chitinase A (ChiA) binds allosamidin with a K-d value of 0.17 +- 0.06 mu M where the main part of the driving force is due to enthalpic change (Delta Hr degrees = 6.2 +- 0.2 kcal/mol) and less to entropic change (-T Delta S-r degrees = 3.2 kcal/mol). A large part of Delta H is due to allosamidin stacking with Trp(167) in the 3 subsite. Binding of allosamidin to both chitinase B (ChiB) (K-d = 0.16 +- 0.04 pM) and chitinase C (ChiC) (K-d = 2.0 +- 0.2 1ilY1) is driven by entropy (Delta H-r degrees = 3.8 0.2 kcal/mol and -T Delta S-r degrees = 13.2 kcal/mol for ChiB and Delta H-r degrees = 0.6 +- 0.1 and T Delta S-r degrees = 7.3 kcal/mol for ChiC). For ChiC, the entropic term is dominated by changes in solvation entropy (Delta S-conf = 37 cal/K center dot mol and Delta S-solv=15 cal/K center dot mol), while, for ChiB, changes in conformational entropy dominate (Delta S-conf = 37 cal/K center dot mol and Delta S-solv = 15 cal/K center dot mol). Corresponding values for ChiA are Delta S-conf = 4 cal/K center dot mol and Delta S-solv = 15 cal/K center dot mol. These remarkable differences in binding parameters reflect the different architectures of the catalytic centers in these enzymes that are adapted to different types of actions: ChiA and ChiB are processive enzymes that move in opposite directions, meaning that allosamidin binds in to "product" subsites in ChiB, while it binds to polymer-binding subsites in ChiA. The values for ChiC are compatible with this enzyme being a nonprocessive endochitinase with a much more open and solvated substrate-binding-site cleft.