. 2012 May 4;8(3):437–502. doi: 10.1007/s11302-012-9309-4

Table 8.

Explanation of the observed effects of previously reported active site mutations of NPPs on activity

Residue^a	Function	Mutant^a	Effect	Explanation	References
D218	Metal 2 binding	D218N (1)	Inactive	Improper cofactor coordination	[367]
K255	Start of first phosphate-binding loop	K255A (1)	Reduced activity	Slight distortion of active site structure, influence on K_m and k_cat/K_m unclear	[367]
T256	Zn²⁺-activated Thr alkoxide acts as nucleophile	T256A (1)	Complete loss of activity	Nucleophilic attack on substrate not possible	[367]
		T256A (2)			[384, 431]
		T256A (7)			[631]
		T256A (wheat NPP)			[454]
		T256S (1)	Almost complete loss of activity, trapping of covalent intermediate	Nucleophilic attack on substrate still possible but distorted active site geometry with potentially larger effect on 2nd catalytic step	[367]
		T256S (wheat NPP)	Complete loss of PDE activity but large gain in activity towards monoester substrates	Thr/Ser alkoxide as major determinant for diesterase vs. monoesterase activity	[454]
F257	Binding of nucleobase/lipid	F257A (1)	Reduced activity	Most likely reduced substrate affinity	[367]
F257	Binding of nucleobase/lipid	F257A (2)	Reduced activity	Most likely reduced substrate affinity	[445]
N277	Phosphate binding, stabilization of transition state	N277A	Complete loss of activity	Missing stabilization of transition state	[445]
		N277A	Strong reduction in activity	Missing stabilization of transition state	[431]
		N277R (wheat NPP)	Complete loss of PDE activity but large gain in activity towards monoester substrates	Positively charged side chain allows monoester cleavage like in APs	[454]
L290	Binding of nucleobase	L290A (2)	Reduced activity	Most likely reduced substrate affinity	[445]
F321	Binding of nucleobase	F321Q (2)	Partial loss of activity	Exact role for nucleotide binding remains unclear	[431]
		F321S (wheat NPP)	Reduction of activity		[454]
		F321A (2)	Reduced PDE activity, unaltered LysoPLD activity		[445]
Y371	Nucleoside binding	Y371A (2)	Reduced activity	Distortion in nucleoside/lipid-binding pocket	[445]
Y371	Nucleoside binding	Y371Q (2)	Reduced activity	Distortion in nucleoside/lipid-binding pocket	[431]
D376	Metal 1 binding	D376N (1)	Largely inactive, trapping of covalent intermediate, slightly rescued by high Zn²⁺	Reduced metal affinity, missing activation of nucleophilic water for second catalytic step	[367]
H380	Metal 1 binding	H380Q (2)	Inactive	Reduced metal affinity	[384]
H380	Metal 1 binding	H380Q (1)	Largely inactive, trapping of covalent intermediate, rescued by high Zn²⁺	Reduced metal affinity	[367]
D423	Metal 2 binding	D423N (1)	Inactive, slightly rescued by high Zn²⁺	Reduced metal affinity	[367]
H424	Metal 2 binding	H424Q (2)	Inactive		[384]
H535	Metal 1 binding	H535Q (1)	Inactive, rescued by high Zn²⁺	Reduced metal affinity	[367]
H535	Metal 1 binding	H535Q (2)	Inactive	Reduced metal affinity	[384]

^aHuman NPP1 residue numbering is used. Numbers in brackets refer to the NPP enzyme used in the study