RESUMO
This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R) confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR) located within the insert could act as an RXR-type endoplasmic reticulum (ER) retention signal. Arginine residues (R) within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E), either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA), radioligand binding assay, bioluminescence resonance energy transfer (BRET²) ß-arrestin 2 (ßarr2) recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E) substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced ßarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3) of the D2L-R appears to be the ER retention signal.
Assuntos
Arginina/química , Arginina/metabolismo , Dopamina/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Isoformas de Proteínas , Ensaio Radioligante , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.
Assuntos
Proteínas de Fluorescência Verde/análise , Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/farmacologia , Medições Luminescentes , Receptor de Insulina/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Células Cultivadas , Humanos , Insulina/análogos & derivados , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.
Assuntos
Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Peptídeos Semelhantes ao Glucagon/farmacologia , Vias Neurais/efeitos dos fármacos , Animais , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/efeitos dos fármacos , Camundongos , RatosRESUMO
This study aimed to functionally characterize ß2-adrenergic (ß2AR) and insulin receptor (IR) heteromers in regard to ß-arrestin 2 (ßarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR ß chain in heteromerization with ß2AR. Evidence for ß2AR:IR:ßarr2 complex formation and the specificity of the IR:ßarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of ß2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-ßarr2 recruitment to the ß2AR:IR complex; the IR:ßarr2 interaction was found to only be constitutive. The constitutive IR:ßarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-ßarr2 1-185 mutant lacking the proposed IR binding domain. ß2AR:IR heteromerization also influenced the pharmacological phenotype of ß2AR, i.e., its efficacy in recruiting ßarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR ß chain in the interaction with ß2AR was provided by BRET2 saturation and HIT assays using an IR 1-1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1-1271-RLuc8:ß2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1-1271 and ß2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of ßarr2 recruitment to the IR 1-1271:ß2AR heteromer. Complementary 3D visualization of ß2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in ß2AR:IR heteromerization. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , beta-Arrestinas/química , beta-Arrestinas/metabolismo , Células HEK293 , Humanos , Receptor Cross-Talk , Transdução de SinaisRESUMO
Insulin-like peptide 3 (INSL3) binds to a G protein-coupled receptor (GPCR) called relaxin family peptide receptor 2 (RXFP2). RXFP2 belongs to the leucine-rich repeat-containing subgroup (LGR) of class A GPCRs. Negative cooperativity has recently been demonstrated in other members of the LGR subgroup. In this work, the kinetics of INSL3 binding to HEK293 cells stably transfected with RXFP2 (HEK293-RXFP2) have been investigated in detail to study whether negative cooperativity occurs and whether this receptor functions as a dimer. Our results show that negative cooperativity is present and that INSL3-RXFP2 binding shows both similarities and differences with insulin binding to the insulin receptor. A dose-response curve for the negative cooperativity of INSL3 binding had a reverse bell shape reminiscent of that seen for the negative cooperativity of insulin binding to its receptor. This suggests that binding of INSL3 may happen in a trans rather than in a cis way in a receptor dimer. Bioluminescence resonance energy transfer (BRET(2)) experiments confirmed that RXFP2 forms constitutive homodimers. Heterodimerization between RXFP2 and RXFP1 was also observed.
Assuntos
Insulina/metabolismo , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Dimerização , Humanos , Medições Luminescentes , Ligação Proteica , Temperatura , TransfecçãoRESUMO
H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET(2). Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.
Assuntos
Ligação Competitiva/fisiologia , Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Relaxina/metabolismo , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo/farmacocinética , Concentração Osmolar , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Peptídeos/fisiologia , Relaxina/química , Relaxina/farmacocinética , Relaxina/fisiologia , Especificidade por Substrato , TemperaturaRESUMO
The glucagon-like peptide-1 receptor agonists (GLP-1RAs) liraglutide and semaglutide reduce cardiovascular risk in type 2 diabetes patients. The mode of action is suggested to occur through modified atherosclerotic progression. In this study, both of the compounds significantly attenuated plaque lesion development in apolipoprotein E-deficient (ApoE-/-) mice and low-density lipoprotein receptor-deficient (LDLr-/-) mice. This attenuation was partly independent of weight and cholesterol lowering. In aortic tissue, exposure to a Western diet alters expression of genes in pathways relevant to the pathogenesis of atherosclerosis, including leukocyte recruitment, leukocyte rolling, adhesion/extravasation, cholesterol metabolism, lipid-mediated signaling, extracellular matrix protein turnover, and plaque hemorrhage. Treatment with semaglutide significantly reversed these changes. These data suggest GLP-1RAs affect atherosclerosis through an anti-inflammatory mechanism.
RESUMO
High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x mL(-1) for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.
Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas/química , Glucana 1,4-alfa-Glucosidase/química , Hordeum/enzimologia , Proteínas de Plantas/química , Proteínas Recombinantes de Fusão/química , Amido/química , alfa-Amilases/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/isolamento & purificação , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , alfa-Amilases/genética , alfa-Amilases/isolamento & purificaçãoRESUMO
Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.
Assuntos
Aspergillus niger/metabolismo , NADP/metabolismo , Via de Pentose Fosfato/fisiologia , Aspergillus niger/enzimologia , Aspergillus niger/genética , Clonagem Molecular , Primers do DNA , Glucosefosfato Desidrogenase/genética , Análise dos Mínimos Quadrados , Modelos Biológicos , Dados de Sequência Molecular , Fosfogluconato Desidrogenase/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Análise de Regressão , Transcetolase/genéticaRESUMO
Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber type-specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese, and type 2 diabetic subjects before and after a hyperinsulinemic-euglycemic clamp. Type I fibers compared with type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), and pyruvate dehydrogenase-E1α (PDH-E1α) and a lower protein content of Akt2, TBC1 domain family member 4 (TBC1D4), and TBC1D1. In type I fibers compared with type II fibers, the phosphorylation response to insulin was similar (TBC1D4, TBC1D1, and GS) or decreased (Akt and PDH-E1α). Phosphorylation responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS, and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared with lean and obese subjects. We conclude that human type I muscle fibers compared with type II fibers have a higher glucose-handling capacity but a similar sensitivity for phosphoregulation by insulin.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Obesidade/metabolismo , Transdução de Sinais/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and ß(2)-adrenergic receptor (ß(2)AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on ß(2)AR properties, we showed that IR coexpression had no effect on the ligand binding properties of ß(2)AR; however, IR reduced ß(2)AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between ß(2)AR and IR, we employed quantitative BRET(2) saturation and competition assays. Saturation assay data suggested constitutive ß(2)AR and IR homo- and heteromerization. Calculated acceptor/donor (AD50) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET(2) signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET(2) data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of ß(2)AR and IR identified intracellular domains encompassing residues at the end of the 7th TM domain and C-terminal tail of ß(2)AR and a cytoplasmic part of the IR ß chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order ß(2)AR:IR oligomer formation, likely comprising heteromers of homodimers.
Assuntos
Biologia Computacional , Modelos Biológicos , Receptor de Insulina/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Algoritmos , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional/métodos , Expressão Gênica , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Receptor de Insulina/química , Receptor de Insulina/genética , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The insulin/relaxin superfamily of peptide hormones comprises 10 members in humans. The three members of the insulin-related subgroup bind to receptor tyrosine kinases (RTKs), while four of the seven members of the relaxin-like subgroup are now known to bind to G-protein-coupled receptors (GPCRs), the so-called relaxin family peptide receptors (RXFPs). Both systems have a long evolutionary history and play a critical role in fundamental biological processes, such as metabolism, growth, survival and longevity, and reproduction. The structural biology and ligand-binding kinetics of the insulin and insulin-like growth factor I receptors have been studied in great detail, culminating in the recent crystal structure of the insulin receptor extracellular domain. Some of the fundamental properties of these receptors, including constitutive dimerization and negative cooperativity, have recently been shown to extend to other RTKs and GPCRs, including RXFPs, confirming kinetic observations made over 30 years ago.
Assuntos
Insulina/química , Insulina/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Relaxina/química , Relaxina/metabolismo , Regulação Alostérica , Humanos , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G-protein-coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone receptors, which like RXFP1 and -2 belong to the leucine-rich repeat-containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Insulina/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Relaxina/metabolismoRESUMO
The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.
Assuntos
Aspergillus niger/enzimologia , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Sequência de Aminoácidos , Aspergillus niger/genética , Fenômenos Biofísicos , Biofísica , Varredura Diferencial de Calorimetria , Sequência de Carboidratos , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Dimerização , Inibidores Enzimáticos/química , Glucana 1,4-alfa-Glucosidase/genética , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Desnaturação Proteica , Estrutura Quaternária de Proteína , Soluções , TemperaturaRESUMO
Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.