Roskoski R, Ritchie P
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119, USA.
Arch Biochem Biophys. 1998 Aug 15;356(2):167-76. doi: 10.1006/abbi.1998.0768.
Protein farnesyltransferase and protein geranylgeranyltransferase-I catalyze the prenylation of a cysteinyl group located four residues upstream of the carboxyl terminus. The identity of the carboxyterminal residue plays a significant role in determining the ability of compounds to bind to each enzyme and to serve as substrate. We compared the binding and substrate specificities of peptides with carboxyterminal substitutions to determine which residues promote selectivity and which residues promote recognition by both enzymes. Using tetrapeptide inhibitors with the general structure l-penicillamine-valine-isoleucine-X and substrates with the structure Lys-Lys-Ser-Ser-Cys-Val-Ile-X, we measured their respective Ki, Km, and kcat values for both recombinant rat protein farnesyltransferase and recombinant rat protein geranylgeranyltransferase-I. We studied the roles of carboxyterminal branched residues (leucine, isoleucine, valine, and penicillamine) and linear residues (methionine, cysteine, homocysteine, alanine, aminobutyrate, and aminohexanoate) in promoting interaction with the enzymes. For protein geranylgeranyltransferase-I, peptide substrates with carboxyterminal branched or linear residues had Km values that were 5- to 15-fold greater than the Ki values of the corresponding peptide inhibitors. For protein farnesyltransferase, peptide substrates with carboxyterminal branched residues, proline, or homoserine had Km values that were 7- to 200-fold greater than the Ki values of the corresponding peptide inhibitors. For protein farnesyltransferase the Km and Ki values for peptides ending with linear residues were in general agreement. Our studies indicate that the substrate and inhibitor binding specificities of protein geranylgeranyltransferase was much more restricted than those of protein farnesyltransferase.
蛋白质法尼基转移酶和蛋白质香叶基香叶基转移酶-I催化位于羧基末端上游四个残基处的半胱氨酰基的异戊二烯化。羧基末端残基的身份在决定化合物与每种酶结合并作为底物的能力方面起着重要作用。我们比较了具有羧基末端取代的肽的结合和底物特异性，以确定哪些残基促进选择性，哪些残基促进两种酶的识别。使用具有通用结构l-青霉胺-缬氨酸-异亮氨酸-X的四肽抑制剂和具有结构赖氨酸-赖氨酸-丝氨酸-丝氨酸-半胱氨酸-缬氨酸-异亮氨酸-X的底物，我们测量了它们对重组大鼠蛋白质法尼基转移酶和重组大鼠蛋白质香叶基香叶基转移酶-I的各自的Ki、Km和kcat值。我们研究了羧基末端支链残基（亮氨酸、异亮氨酸、缬氨酸和青霉胺）和线性残基（甲硫氨酸、半胱氨酸、高半胱氨酸、丙氨酸、氨基丁酸和氨基己酸）在促进与酶相互作用中的作用。对于蛋白质香叶基香叶基转移酶-I，具有羧基末端支链或线性残基的肽底物的Km值比相应肽抑制剂的Ki值大5至15倍。对于蛋白质法尼基转移酶，具有羧基末端支链残基、脯氨酸或高丝氨酸的肽底物的Km值比相应肽抑制剂的Ki值大7至200倍。对于蛋白质法尼基转移酶，以线性残基结尾的肽的Km和Ki值总体上一致。我们的研究表明，蛋白质香叶基香叶基转移酶的底物和抑制剂结合特异性比蛋白质法尼基转移酶的受限制得多。
